Dietary Supplement Laboratory 

Quality Assurance Program: 

Exercise L Final Report

Melissa M. Phillips

Catherine A. Rimmer

Laura J. Wood

This publication is available free of charge from:

https://doi.org/10.6028/NIST.IR.8154

NIST IR 8154
NIST IR 8154

Dietary Supplement Laboratory 

Quality Assurance Program: 

Exercise L Final Report

Melissa M. Phillips

Catherine A. Rimmer

Laura J. Wood 

Chemical Sciences Division

Material Measurement Laboratory 

This publication is available free of charge from:

https://doi.org/10.6028/NIST.IR.8154

October 2016

U.S. Department of Commerce 

Penny Pritzker, Secretary

National Institute of Standards and Technology

Willie May, Under Secretary of Commerce for Standards and Technology and Director
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TABLE OF CONTENTS

ABSTRACT ..................................................................................................................................1

INTRODUCTION..........................................................................................................................1

OVERVIEW OF DATA TREATMENT AND REPRESENTATION .....................................2

Statistics ...............................................................................................................................2

Individualized Data Table ....................................................................................................3

Summary Data Table ...........................................................................................................4

Graphs ..................................................................................................................................4

Data Summary View (Method Comparison Data Summary View) ........................4

Sample/Sample Comparison View ..........................................................................5

NUTRITIONAL ELEMENTS (Iodine) IN CAT FOOD AND MULTIVITAMIN 

TABLETS ...............................................................................................................6

Study Overview ...................................................................................................................6

Sample Information .............................................................................................................6

Cat Food ...................................................................................................................6

Multivitamin ............................................................................................................6

Study Results ......................................................................................................................7

Technical Recommendations ...............................................................................................7

Table 1. Individualized data summary table (NIST) for iodine in cat food and 

multivitamin tablets .................................................................................................8

Table 2. Data summary table for iodine in cat food and multivitamin 

tablets .......................................................................................................................9

Figure 1. Iodine in SRM 3290 Dry Cat Food (data summary view – digestion and analytical 

method) ..................................................................................................................10

Figure 2. Iodine in SRM 3280 Multivitamin/Multielement Tablets (data summary view – 

digestion and analytical method) ...........................................................................11

Figure 3. Iodine in SRM 3290 Dry Cat Food and SRM 3280 Multivitamin/Multielement 

Tablets (sample/sample comparison view) ............................................................12

TOXIC ELEMENTS (As and Pb) IN ST. JOHN’S WORT DIETARY SUPPLEMENTS ..13

Study Overview .................................................................................................................13

Sample Information ...........................................................................................................13

St. John’s Wort Aerial Parts...................................................................................13

St. John’s Wort Methanol Extract ..........................................................................13

Study Results ....................................................................................................................14

Technical Recommendations .............................................................................................14

Table 3. Individualized data summary table (NIST) for arsenic and lead in St. John’s wort 

dietary supplements ...............................................................................................16

Table 4. Data summary table for arsenic in St. John’s wort dietary supplements ...........17

Table 5. Data summary table for lead in St. John’s wort dietary supplements ................18
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Figure 4. Arsenic in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts 

(data summary view – digestion and analytical method) .......................................19

Figure 5. Arsenic in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol 

Extract (data summary view – digestion and analytical method) ..........................20

Figure 6. Lead in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data 

summary view – digestion and analytical method) ................................................21

Figure 7. Lead in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract 

(data summary view – digestion and analytical method) .......................................22

Figure 8. Arsenic in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts 

and SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract

(sample/sample comparison view) .........................................................................23

Figure 9. Lead in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and 

SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract

(sample/sample comparison view) .........................................................................24

WATER-SOLUBLE VITAMINS (Biotin) IN DIETARY SUPPLEMENTS .........................25

Study Overview .................................................................................................................25

Sample Information ...........................................................................................................25

Cat Food .................................................................................................................25

Multivitamin ..........................................................................................................25

Study Results .....................................................................................................................25

Technical Recommendations .............................................................................................26

Table 6. Individualized data summary table (NIST) for biotin in dietary supplements ..28

Table 7. Data summary table for biotin in dietary supplements ......................................29

Figure 10. Biotin in SRM 3290 Dry Cat Food (data summary view – sample preparation 

and analytical method) ...........................................................................................30

Figure 11. Biotin in SRM 3280 Multivitamin/Multielement Tablets (data summary view – 

sample preparation and analytical method) ...........................................................31

Figure 12. Biotin in SRM 3290 Dry Cat Food and SRM 3280 Multivitamin/Multielement 

Tablets (sample/sample comparison view) ............................................................32

XANTHOPHYLLS (Lutein and Zeaxanthin) IN DIETARY SUPPLEMENTS ...................33

Study Overview .................................................................................................................33

Sample Information ...........................................................................................................33

Spinach .......................................................................................................................

Multivitamin/Multielement Tablets .......................................................................33

Study Results .....................................................................................................................34

Technical Recommendations .............................................................................................34

Table 8. Individualized data summary table (NIST) for lutein and zeaxanthin in dietary 

supplements............................................................................................................36

Table 9. Data summary table for lutein in dietary supplements ......................................37

Table 10. Data summary table for zeaxanthin in dietary supplements ............................38

Figure 13. Lutein in SRM 2385 Slurried Spinach (data summary view – sample preparation

and analytical method) ...........................................................................................39

Figure 14. Lutein in SRM 3280 Multivitamin/Multielement Tablets (data summary view 

– sample preparation and analytical method) ........................................................40
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Figure 15. Zeaxanthin in SRM 2385 Slurried Spinach (data summary view – sample 

preparation and analytical method) ........................................................................41

Figure 16. Zeaxanthin in SRM 3280 Multivitamin/Multielement Tablets (data summary 

view – sample preparation and analytical method) ................................................42

Figure 17. Lutein in SRM 2385 Slurried Spinach and SRM 3280 

Multivitamin/Multielement Tablets (sample/sample comparison view) ...............43

Figure 18. Zeaxanthin in SRM 2385 Slurried Spinach and SRM 3280 

Multivitamin/Multielement Tablets (sample/sample comparison view) ...............44

FATTY ACIDS IN FISH OILS ..................................................................................................45

Study Overview .................................................................................................................45

Sample Information ...........................................................................................................45

Fish Oil 1................................................................................................................45

Fish Oil 2................................................................................................................45

Study Results .....................................................................................................................46

Technical Recommendations .............................................................................................47

Table 11. Individualized data summary table (NIST) for fatty acids in fish oils ............48

Table 12. Data summary table for linoleic acid in fish oils .............................................49

Table 13. Data summary table for α-linolenic acid in fish oils ........................................50

Table 14. Data summary table for γ-linolenic acid in fish oils ........................................51

Table 15. Data summary table for arachidonic acid in fish oils ......................................52

Table 16. Data summary table for EPA in fish oils .........................................................53

Table 17. Data summary table for DHA in fish oils ........................................................54

Figure 19. Linoleic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil 

(data summary view – sample preparation and analytical method) .......................55

Figure 20. Linoleic acid in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil 

(data summary view – sample preparation and analytical method) .......................56

Figure 21. α-Linolenic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil 

(data summary view – sample preparation and analytical method) .......................57

Figure 22. α-Linolenic acid in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish 

Oil (data summary view – sample preparation and analytical method) .................58

Figure 23. γ-Linolenic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil 

(data summary view – sample preparation and analytical method) .......................59

Figure 24. γ-Linolenic acid in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish 

Oil (data summary view – sample preparation and analytical method) .................60

Figure 25. Arachidonic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish 

Oil (data summary view – sample preparation and analytical method) .................61

Figure 26. Arachidonic acid in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish 

Oil (data summary view – sample preparation and analytical method) .................62

Figure 27. EPA in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil (data 

summary view – sample preparation and analytical method) ................................63

Figure 28. EPA in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (data 

summary view – sample preparation and analytical method) ................................64

Figure 29. DHA in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil (data 

summary view – sample preparation and analytical method) ................................65
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Figure 30. DHA in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (data 

summary view – sample preparation and analytical method) ................................66

Figure 31. Linoleic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil

and SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (sample/sample 

comparison view) ...................................................................................................67

Figure 32. α-Linolenic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil

and SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (sample/sample 

comparison view) ...................................................................................................68

Figure 33. γ-Linolenic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil

and SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (sample/sample 

comparison view) ...................................................................................................69

Figure 34. Arachidonic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish 

Oil and SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil 

(sample/sample comparison view) .........................................................................70

Figure 35. EPA in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil and SRM 

3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (sample/sample 

comparison view) ...................................................................................................71

Figure 36. DHA in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil and SRM 

3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (sample/sample 

comparison view) ...................................................................................................72

CHLOROGENIC ACID, FLAVONOIDS, AND NAPHTHODIANTHRONES IN 

ST JOHN’S WORT DIETARY SUPPLEMENTS ...........................................73

Study Overview .................................................................................................................73

Sample Information ...........................................................................................................73

St. John’s Wort Aerial Parts...................................................................................73

St. John’s Wort Methanol Extract ..........................................................................73

Study Results .....................................................................................................................74

Technical Recommendations .............................................................................................75

Table 18. Individualized data summary table (NIST) for chlorogenic acid, flavonoids, and 

naphthodianthrones in St. John’s wort dietary supplements. .................................76

Table 19. Data summary table for chlorogenic acid in St. John’s wort dietary supplements

................................................................................................................................77

Table 20. Data summary table for rutin in St. John’s wort dietary supplements .............78

Table 21. Data summary table for hyperoside in St. John’s wort dietary supplements ...79

Table 22. Data summary table for isoquercitrin in St. John’s wort dietary supplements 80

Table 23. Data summary table for quercitrin in St. John’s wort dietary supplements .....81

Table 24. Data summary table for quercetin in St. John’s wort dietary supplements .....82

Table 25. Data summary table for amentoflavone in St. John’s wort dietary 

supplements............................................................................................................83

Table 26. Data summary table for pseudohypericin in St. John’s wort dietary supplements

................................................................................................................................84

Table 27. Data summary table for hypericin in St. John’s wort dietary supplements .....85

Figure 37. Chlorogenic acid in SRM 3262 St. John’s Wort (Hypericum perforatum L.) 

Aerial Parts (data summary view – sample preparation and analytical method) ...86
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Figure 38. Chlorogenic acid in SRM 3264 St. John’s Wort (Hypericum perforatum L.) 

Methanol Extract (data summary view – sample preparation and analytical method)

................................................................................................................................87

Figure 39. Rutin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts

(data summary view – sample preparation and analytical method) .......................88

Figure 40. Rutin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol 

Extract (data summary view – sample preparation and analytical method) ..........89

Figure 41. Hyperoside in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial 

Parts (data summary view – sample preparation and analytical method) ..............90

Figure 42. Hyperoside in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol 

Extract (data summary view – sample preparation and analytical method) ..........91

Figure 43. Isoquercitrin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial 

Parts (data summary view – sample preparation and analytical method) ..............92

Figure 44. Isoquercitrin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) 

Methanol Extract (data summary view – sample preparation and analytical method)

................................................................................................................................93

Figure 45. Quercitrin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial 

Parts (data summary view – sample preparation and analytical method) ..............94

Figure 46. Quercitrin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol 

Extract (data summary view – sample preparation and analytical method) ..........95

Figure 47. Quercetin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial 

Parts (data summary view – sample preparation and analytical method) ..............96

Figure 48. Quercetin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol 

Extract (data summary view – sample preparation and analytical method) ..........97

Figure 49. Amentoflavone in St. John’s Wort (Hypericum perforatum L.) Aerial Parts

(data summary view – sample preparation and analytical method) .......................98

Figure 50. Amentoflavone in SRM 3264 St. John’s Wort (Hypericum perforatum L.) 

Methanol Extract (data summary view – sample preparation and analytical method)

................................................................................................................................99

Figure 51. Pseudohypericin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) 

Aerial Parts (data summary view – sample preparation and analytical method) .100

Figure 52. Pseudohypericin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) 

Methanol Extract (data summary view – sample preparation and analytical method)

..............................................................................................................................101

Figure 53. Hypericin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial 

Parts (data summary view – sample preparation and analytical method) ............102

Figure 54. Hypericin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol 

Extract (data summary view – sample preparation and analytical method) ........103

Figure 55. Chlorogenic acid in SRM 3262 St. John’s Wort (Hypericum perforatum L.) 

Aerial Parts and SRM 3264 Hypericum perforatum (St. John’s Wort) Methanol

Extract (sample/sample comparison view) ............................................................104

Figure 56. Rutin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts

and SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract

(sample/sample comparison view) .........................................................................105
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Figure 57. Hyperoside in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial 

Parts and SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract

(sample/sample comparison view) .........................................................................106

Figure 58. Isoquercitrin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial 

Parts and SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract

(sample/sample comparison view) .........................................................................107

Figure 59. Quercitrin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial 

Parts and SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract

(sample/sample comparison view) .........................................................................108

Figure 60. Quercetin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts

and SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract

(sample/sample comparison view) .........................................................................109

Figure 61. Amentoflavone in SRM 3262 St. John’s Wort (Hypericum perforatum L.) 

Aerial Parts and SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol 

Extract (sample/sample comparison view) ............................................................110

Figure 62. Pseudohypericin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) 

Aerial Parts and SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol 

Extract (sample/sample comparison view) ............................................................111

Figure 63. St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. 

John’s Wort (Hypericum perforatum L.) Methanol Extract (sample/sample

comparison view) .................................................................................................112
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ABSTRACT

The NIST Dietary Supplement Laboratory Quality Assurance Program (DSQAP) was established 

in collaboration with the National Institutes of Health (NIH) Office of Dietary Supplements (ODS) 

in 2007 to enable members of the dietary supplements community to improve the accuracy of 

measurements for demonstration of compliance with various regulations including the dietary 

supplement current good manufacturing practices (cGMPs). Exercise L of this program offered 

the opportunity for laboratories to assess their in-house measurements of nutritional elements

(iodine), contaminants (lead and arsenic), water-soluble vitamins (biotin), fat-soluble vitamins 

(lutein and zeaxanthin), fatty acids (omega-3 and -6), and botanical marker compounds 

(chlorogenic acid, flavonoids, and naphthodianthrones) in foods and/or botanical dietary 

supplement ingredients and finished products. 

INTRODUCTION

The dietary supplement industry in the US is booming, with two-thirds of adults considering 

themselves to be supplement users.1

 Consumption of dietary supplements, which includes vitamin 

and mineral supplements, represents an annual US expenditure of more than $40 billion. These 

figures represent an increasing American and worldwide trend, and as a result, it is critically 

important that both the quality and safety of these products are verified and maintained. 

The Dietary Supplement Health and Education Act of 1994 (DSHEA) amended the Federal Food, 

Drug, and Cosmetic Act to create the regulatory category called dietary supplements. The DSHEA 

also gave the FDA authority to write current Good Manufacturing Practices (cGMPs) that require 

manufacturers to evaluate the identity, purity, and composition of their ingredients and finished 

products. In addition, the DSHEA authorized the establishment of the Office of Dietary

Supplements at the National Institutes of Health (NIH ODS). To enable members of the dietary 

supplements community to improve the accuracy of the measurements required for compliance 

with these and other regulations, NIST established the Dietary Supplement Laboratory Quality 

Assurance Program (DSQAP) in collaboration with the NIH ODS in 2007. 

The program offers the opportunity for laboratories to assess their in-house measurements of active 

or marker compounds, nutritional elements, contaminants (toxic elements, pesticides, 

mycotoxins), and fat- and water-soluble vitamins in foods as well as botanical dietary supplement 

ingredients and finished products. Reports and certificates of participation are provided and can 

be used to demonstrate compliance with the cGMPs. In addition, NIST and the DSQAP assist the 

ODS Analytical Methods and Reference Materials program (AMRM) at the NIH in supporting the 

development and dissemination of analytical tools and reference materials. In the future, results 

from DSQAP exercises could be used by ODS to identify problematic matrices and analytes for 

which an AOAC INTERNATIONAL Official Method of Analysis would benefit the dietary 

supplement community. 

NIST has experience in the administration of quality assurance programs, but the DSQAP takes a 

unique approach. In other NIST quality assurance programs, a set of analytes is measured 

1 Walsh, T. (2012) Supplement Usage, Consumer Confidence Remain Steady According to New Annual Survey from 

CRN. Council for Responsible Nutrition, Washington, DC.
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repeatedly over time in the same or similar matrices to demonstrate and improve laboratory 

performance. In contrast, the wide range of matrices and analytes under the “dietary supplement” 

umbrella means that not every laboratory is interested in every sample or analyte. The constantly 

changing dietary supplement market, and the enormous diversity of finished products, makes 

repeated determination of a few target compounds in a single matrix of little use to participants. 

Instead, participating laboratories are interested in testing in-house methods on a wide variety of 

challenging, real-world matrices to demonstrate that their performance is comparable to that of the 

community and that their methods provide accurate results. In an area where there are few standard 

methods, the DSQAP offers a unique tool for assessment of the quality of measurements, provides 

feedback about performance, and can assist participants in improving laboratory operations.

This report summarizes the results from the eleventh exercise of the DSQAP, Exercise L. Eightytwo laboratories responded to the call for participants distributed in October 2015. Samples were 

shipped to participants in January 2016, and results were returned to NIST by March 2016. This 

report contains the final data and information that was disseminated to the participants in October 

2016.

OVERVIEW OF DATA TREATMENT AND REPRESENTATION

Individualized data tables and certificates are provided to the participants that have submitted data 

in each study, in addition to this report. Examples of the data tables using NIST data are also 

included in each section of this report. Community tables and graphs are provided using 

randomized laboratory codes, with identities known only to NIST and individual laboratories. The 

statistical approaches are outlined below for each type of data representation.

Statistics

Data tables and graphs throughout this report contain information about the performance of each 

laboratory relative to that of the other participants in this study and relative to a target around the 

expected result, if available. All calculations are performed in PROLab Plus (QuoData GmbH, 

Dresden, Germany).2 The consensus mean and standard deviation are calculated according to the 

robust algorithm outlined in ISO 13528:2015(E), Annex C.3 The algorithm is summarized here in 

simplified form.

Initial values of the consensus mean, x*, and consensus standard deviation, s*, are estimated as

x* = median of xi (i = 1, 2,...,n)

s* = 1.483 × median of |xi – x*| (i = 1, 2,...,n).

These initial values for x* and s* are updated by first calculating the expanded standard deviation, 

δ, as

δ = 1.5 × s*.

 2 Certain commercial equipment, instruments or materials are identified in this certificate to adequately specify the experimental 

procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and 

Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

3 ISO 13528:2015(E), Statistical methods for use in proficiency testing by interlaboratory comparisons, pp. 53-54.
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Then each xi is compared to the expanded range and adjusted to xi* as described below to reduce 

the effect of outliers.

If xi < x* – δ, then xi* = x* – δ.

If xi > x* + δ, then xi* = x* + δ.

Otherwise, xi* = xi.

New values of x*, s*, and δ are calculated iteratively until the process converges. Convergence is 

taken as no change from one iteration to the next in the third significant figure of s* and in the 

equivalent digit in x*:

x* = ∑ xxii nn ∗ ii=1

nn

s* = 1.134 × �∑ �xxii

∗−xx∗� nn

ii=1

nn−1 .

Individualized Data Table

The data in this table is individualized to each participating laboratory and is provided to allow 

participants to directly compare their data to the summary statistics (consensus or community data 

as well as NIST certified, reference, or estimated values). The upper left of the data table includes 

the randomized laboratory code. Tables included in this report are generated using NIST data to 

protect the identity and performance of participants.

Section 1 of the data table contains the laboratory results as reported, including the mean and 

standard deviation when multiple values were reported. A blank indicates that NIST does not have 

data on file for that laboratory for a particular analyte or matrix. An empty box for standard

deviation indicates that only a single value was reported and therefore that value was not included 

in the calculation of the consensus data.3

Also in Section 1 are two Z-scores. The first Z-score, Z’comm, is calculated with respect to the 

community consensus value, taking into consideration bias that may result from the uncertainty in 

the assigned consensus value, using x* and s*:

ZZ′cccccccc = xxii−xx∗

√2ss∗

The second Z-score, ZNIST, is calculated with respect to the target value (NIST certified, reference, 

or estimated value), using xNIST and U95 (the expanded uncertainty) or sNIST (the standard deviation 

of NIST measurements):

ZZNNNNNNNN = xxii−xxNNNNNNNN

UU95

or

ZZNNNNNNNN = xxii−xxNNNNNNNN

ssNNNNNNNN

.
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The significance of the Z-score is as follows:

• |Z| < 2 indicates that the laboratory result is considered to be within the community 

consensus range (for Z’comm) or NIST target range (for ZNIST).

• 2 < |Z| < 3 indicates that the laboratory result is considered to be marginally different from 

the community consensus value (for Z’comm) or NIST target value (for ZNIST).

• |Z| > 3 indicates that the laboratory result is considered to be significantly different from 

the community consensus value (for Z’comm) or NIST target value (for ZNIST).

Section 2 of the data table contains the community results, including the number of laboratories 

reporting more than a single value for a given analyte1, the mean value determined for each analyte, 

and a robust estimate of the standard deviation of the reported values.4 Consensus means and 

standard deviations are calculated using the laboratory means; if a laboratory reported a single 

value, the reported value is not included.3 Additional information on calculation of the consensus 

mean and standard deviation can be found in the previous section.

Section 3 of the data table contains the target values for each analyte. When possible, the target 

value is a certified or reference value determined at NIST. Certified values and the associated 

expanded uncertainty (U95) have been determined with two independent analytical methods at 

NIST, by collaborating laboratories, or in some combination. Reference values are assigned using 

NIST values obtained from the average and standard deviation of measurements made using a 

single analytical method or by measurements obtained from collaborating laboratories. For both 

certified and reference values, at least six samples have been tested and duplicate preparations 

from the sample package have been included, allowing the uncertainty to encompass variability 

due to inhomogeneity within and between packages. For samples in which a NIST certified or 

reference value is not available, the analytes are measured at NIST using an appropriate method. 

The NIST-assessed value represents the mean of at least three replicates. For materials acquired 

from another proficiency testing program, the consensus value and uncertainty from the completed 

round is used as the target range.

Summary Data Table

This data table includes a summary of all reported data for a particular analyte in a particular study. 

Participants can compare the raw data for a single laboratory to data reported by the other 

participating laboratories or to the consensus data. A blank indicates that the laboratory signed up 

and received samples for that particular analyte and matrix, but NIST does not have data on file 

for that laboratory.

Graphs

Data Summary View (Method Comparison Data Summary View)

In this view, individual laboratory data are plotted with the individual laboratory standard 

deviation (error bars). Laboratories reporting values below the method quantitation limit are 

shown in this view as downward triangles beginning at the limit of quantitation (LOQ). 

Laboratories reporting values as “below LOQ” can still be successful in the study if the target 

value is also below the laboratory LOQ. The black solid line represents the consensus mean, and 

 4 ISO 13528:2015(E), Statistical methods for use in proficiency testing by interlaboratory comparisons, Annex C.
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the green shaded area represents the consensus variability. Where appropriate, two consensus 

means may be calculated for the same sample if bimodality is identified in the data. In this case, 

two consensus means and ranges will be displayed in the data summary view. The red shaded 

region represents the target zone for “acceptable” performance, which encompasses the NIST 

certified, reference, or estimated value bounded by twice its uncertainty (U95) or standard 

deviation. For the purpose of the DSQAP, a target range spanning twice the uncertainty in the 

NIST value is selected because participants are only asked to make a limited number of 

observations. The size of the y-axis on the data summary view graph represents the range of 

tolerance (values that result in an acceptable Z’ score, |ZZ′| ≤ 2). In this view, the relative locations 

of individual laboratory data and consensus zones with respect to the target zone can be compared 

easily. In most cases, the target zone and the consensus zone overlap, which is the expected result. 

The major program goals are to reduce the size of the consensus zone and center the consensus 

zone about the target value. Analysis of an appropriate reference material as part of a quality 

control scheme can help to identify sources of bias for laboratories reporting results that are 

significantly different from the target zone. In the case in which a method comparison is relevant, 

different colored data points may be used to indicate laboratories that used a specific approach to 

sample preparation, analysis, or quantitation.

Sample/Sample Comparison View

In this view, the individual laboratory results for one sample (NIST SRM with a certified or 

reference value) are compared to the results for another sample (another NIST SRM with a more 

challenging matrix, a commercial sample, etc.). The solid red box represents the target zone for 

the first sample (x-axis) and the second sample (y-axis). The dotted blue box represents the 

consensus zone for the first sample (x-axis) and the second sample (y-axis). The axes of this graph 

are centered about the consensus mean values for each sample or control, to a limit of twice the 

range of tolerance (values that result in an acceptable Z’ score, |ZZ′| ≤ 2). Depending on the 

variability in the data, the axes may be scaled proportionally to better display the individual data 

points for each laboratory. In some cases, when the consensus and target ranges have limited 

overlap, the solid red box may only appear partially on the graph. If the variability in the data is 

high (greater than 100 % relative standard deviation (RSD)), the dotted blue box may also only 

appear partially on the graph. This view emphasizes trends in the data that may indicate potential 

calibration issues or method biases. One program goal is to identify such calibration or method 

biases and assist participants in improving analytical measurement capabilities. In some cases, 

when two equally challenging materials are provided, the same view (sample/sample comparison) 

can be helpful in identifying commonalities or differences in the analysis of the two materials.
6

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NUTRITIONAL ELEMENTS (IODINE) IN CAT FOOD AND MULTIVITAMIN 

TABLETS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 3290 Dry Cat Food and 

SRM 3280 Multivitamin/Multielement Tablets. Participants were asked to use in-house analytical 

methods to determine the mass fraction of iodine in each of the matrices and report values on an 

as-received basis.

Sample Information

Cat Food. Participants were provided with one packet containing approximately 10 g of dry cat 

food. The cat food was blended, aliquotted, and heat-sealed inside 4 mil polyethylene bags, which 

were then sealed inside nitrogen-flushed aluminized plastic bags along with two packets of silica 

gel. Before use, participants were instructed to thoroughly mix the contents of each packet and to 

use a sample size of at least 0.5 g. Participants were asked to store the material at controlled room 

temperature, 10 °C to 30 °C, and to prepare three samples and report three values from the single 

packet provided. Approximate analyte levels were not reported to participants prior to the study. 

The reference value for iodine in SRM 3290 Dry Cat Food was determined at NIST using 

inductively coupled plasma mass spectrometry (ICP-MS) and instrumental neutron activation 

analysis (INAA). The reference values and uncertainties for iodine are provided in the table below, 

both on a dry-mass basis and on an as-received basis accounting for moisture of the material 

(4.36 %).

Reference Mass Fraction in SRM 3290 (mg/kg)

Analyte (dry-mass basis) (as-received basis)

Iodine (I) 3.38 ± 0.54 3.23 ± 0.52

Multivitamin. Participants were provided with one bottle containing 30 multivitamin/multielement 

tablets. Before use, participants were instructed to grind all tablets together and mix the resulting 

powder thoroughly, and to use a sample size of at least 0.5 g. Participants were asked to store the 

material at controlled room temperature, 10 °C to 30 °C, and to prepare three samples and report 

three values from the single bottle provided. Approximate analyte levels were not reported to 

participants prior to the study. The certified value for iodine in SRM 3280 Multivitamin/

Multielement Tablets was determined at NIST using inductively coupled plasma mass 

spectrometry (ICP-MS) and instrumental neutron activation analysis (INAA). The certified values 

and uncertainties for iodine are provided in the table below, both on a dry-mass basis and on an 

as-received basis accounting for moisture of the material (1.37 %).

Certified Mass Fraction in SRM 3280 (mg/kg)

Analyte (dry-mass basis) (as-received basis)

Iodine (I) 132.7 ± 6.6 130.9 ± 6.5
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Study Results

• Thirty-three laboratories enrolled in this exercise and received samples. Eleven 

laboratories reported results for the multivitamin sample (33 % participation). Twelve

laboratories reported results for the cat food sample (36 % participation). Ten and 11 

laboratories were used, respectively, for calculation purposes, see Statistics, page 3.

• The consensus means for iodine in both materials were within the target range with 

acceptable between-laboratory variability (15 % to 20 % RSD).

• A majority of the laboratories reported using hot block digestion (33 %), microwave 

digestion (25 %), or solvent extraction (25 %) for sample preparation. The remaining 

laboratories reported using base hydrolysis or dry ashing, although no values were reported 

by the laboratory that reported using dry ashing.

• A majority of the laboratories reported using ICP-MS (69 %) as their analytical method.

The remaining laboratories reported using ion-selective electrode, ion chromatography 

with conductivity detection, liquid chromatography, and thiosulfate titration, although no 

values were reported by the laboratory that reported using thiosulfate titration.

Technical Recommendations

The following recommendations are based on results obtained from the participants in this study.

• The iodine study had the lowest enrollment (40 %) and participation rate (33 % to 36 %) of a 

nutritional elements study in the last five years. The nutritional elements studies are normally 

some of the most popular with the highest number of participants.

• Over the past five years, nutritional elements studies have had 47 % to 65 % of total 

laboratories enrolled, with 65 % to 83 % participation.

• The low participation in this study could be the result of a lack of interest in iodine or the 

greater challenge posed by analysis of iodine compared to other nutritional elements.

• With a small number of laboratories reporting data, identification of strong trends in the data 

based on the information reported by participants is difficult. The data suggest that ICP-MS 

and digestion sample preparations, acid or base, were slightly more successful than 

chromatography methods and solvent extractions.

• Some suggestions regarding iodine sample preparation are provided below.

• Iodine is a volatile element and can form hydrogen iodide (HI) during acid digestion; care 

must be taken to retain iodine during sample preparation.

• Iodine is also light sensitive and at some stages of sample preparation solutions may need 

to be kept in amber or covered samples vessels.

• When using ICP-MS, an acidic sample solution can result in sample carryover. Using a 

basic solution or a surfactant such as Triton X-100 will improve washout of iodine. Some 

protocols use an alkaline digestion with tetramethylammonium hydroxide (TMAH), but 

extreme caution must be taken when using TMAH, which is a very strong base with high 

toxicity. A safer alternative may be to use an acid digestion then solutions can be 

neutralized with a base such as ammonium hydroxide.

• During sample preparation, iodine can adhere to TFM vessels, so PFA vessels or 

quartz/glass vessels are recommended to eliminate erratic results.
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Table 1. Individualized data summary table (NIST) for iodine in cat food and multivitamin tablets.

Lab Code: NIST

Analyte Sample Units xi si Z'comm ZNIST N x* s* xNIST U 95

Iodine Multivitamin mg/kg 131 13 0.0 10 131 20 131 13

Iodine Cat Food mg/kg 3.2 1.0 0.0 11 3.5 0.7 3.2 1.0

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U 95 ±95% confidence interval

Z'comm Z'-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

National Institute of Standards & Technology

Exercise L - October 2015 - Iodine

1. Your Results 2. Community Results 3. Target
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Table 2. Data summary table for iodine in multivitamin tablets and cat food.

Lab A B C Avg SD A B C Avg SD

NIST 131 13 3.23 1.03

L101

L102

L103 117 93 105 17 3.03 3.34 3.19 0.22

L104

L105

L107 129 139 140 136 6 3.66 3.80 4.69 4.05 0.56

L108

L110

L112

L115 126 123 123 124 2 3.52 3.61 3.45 3.53 0.08

L117

L118 142 141 140 141 1 3.46 3.49 3.29 3.41 0.11

L123 122 129 143 131 11 3.39 2.64 2.68 2.90 0.42

L124

L126

L129 4.25 3.74 4.13 4.04 0.27

L130

L136

L137 202 204 207 205 2 28.72 32.15 30.65 30.51 1.72

L139 101 112 97 103 8 3.06 2.58 2.81 2.82 0.24

L140

L141

L148

L151

L152 140 140 3.90 3.90

L155

L157

L159

L160 138 161 145 148 12 3.30 3.50 3.50 3.43 0.12

L165

L170

L171

L172

L176

L177

L179 133 132 128 131 3 0.72 0.61 0.76 0.70 0.08

L182 121 125 125 124 2 3.88 3.98 4.01 3.96 0.07

 Consensus Mean 131 Consensus Mean 3.52

 Consensus Standard Deviation 20 Consensus Standard Deviation 0.70

 Maximum 205 Maximum 30.51

 Minimum 103 Minimum 0.70

 N 10 N 11

Iodine

SRM 3280 MultivitaminTablets (mg/kg) SRM 3290 Dry Cat Food (mg/kg)

Community 

Results Individual Results
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Figure 1. Iodine in SRM 3290 Dry Cat Food (data summary view – digestion and analytical method). In this view, individual laboratory 

data are plotted (circles) with the individual laboratory standard deviation (rectangle). The color of the data point represents the sample 

preparation (digestion) procedure and analytical method employed. The black solid line represents the consensus mean, and the green

shaded region represents the range of tolerance, calculated as the values above and below the consensus mean that result in an acceptable 

Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, which encompasses the NIST 

reference value bounded by twice its uncertainty (U95).
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Figure 2. Iodine in SRM 3280 Multivitamin/Multielement Tablets (data summary view – digestion and analytical method). In this 

view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The color of the data 

point represents the sample preparation (digestion) procedure and analytical method employed. The black solid line represents the 

consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and below the consensus 

mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, 

which encompasses the NIST certified value bounded by twice its uncertainty (U95).
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Figure 3. Iodine in SRM 3290 Dry Cat Food and SRM 3280 Multivitamin/Multielement Tablets (sample/sample comparison view). 

In this view, the individual laboratory results for one sample (multivitamin) are compared to the results for a second sample (cat food). 

The solid red box represents the target zone for the two samples, multivitamin (x-axis) and cat food (y-axis). The dotted blue box 

represents the consensus zone for multivitamin (x-axis) and cat food (y-axis).
13

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TOXIC ELEMENTS (As AND Pb) IN ST. JOHN’S WORT DIETARY SUPPLEMENTS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 3262 St. John’s Wort

(Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort (Hypericum perforatum

L.) Methanol Extract. Participants were asked to use in-house analytical methods to determine the 

mass fractions of total arsenic (As) and lead (Pb) in each of the matrices and report values on an 

as-received basis. 

Sample Information

St. John’s Wort Aerial Parts. Participants were provided with three packets containing 

approximately 3.3 g of dried St. John’s Wort aerial parts. The dried leaves were ground, 

homogenized, and packaged inside 4 mil polyethylene bags, which were then sealed inside 

nitrogen-flushed aluminized plastic bags along with two packets of silica gel. Before use, 

participants were instructed to thoroughly mix the contents of each packet and use a sample size 

of at least 1.0 g. Participants were asked to store the material at controlled room temperature, 

10 °C to 30 °C, and to report a single value from each packet provided. Approximate analyte 

levels were not reported to participants prior to the study. The target value for arsenic in 

SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts was determined at NIST using 

inductively coupled plasma mass spectrometry (ICP-MS) and instrumental neutron activation 

analysis (INAA). The target value for lead in SRM 3262 St. John’s Wort (Hypericum perforatum 

L.) Aerial Parts was determined at NIST using isotope dilution inductively coupled plasma mass 

spectrometry (ID-ICP-MS). The NIST-determined values and uncertainties for As and Pb are 

provided in the table below, on an as-received basis.

NIST-Determined Mass Fraction in SRM 3262 (ng/g)

Analyte (as-received basis)

Arsenic (As) 145 ± 13

Lead (Pb) 933 ± 137

St. John’s Wort Methanol Extract. Participants were provided with three packets containing 

approximately 1.6 g of St. John’s Wort methanol extract. The extract was ground, homogenized, 

and packaged inside 4 mil polyethylene bags, which were then sealed inside nitrogen-flushed 

aluminized plastic bags along with two packets of silica gel. Before use, participants were 

instructed to thoroughly mix the contents of each packet and use a sample size of at least 0.6 g. 

Participants were asked to store the material at controlled room temperature, 10 °C to 30 °C, and 

to report a single value from each packet provided. Approximate analyte levels were not reported 

to participants prior to the study. The target value for arsenic in SRM 3264 St. John’s Wort

(Hypericum perforatum L.) Methanol Extract was determined at NIST using inductively coupled 

plasma mass spectrometry (ICP-MS). The certified value for lead in SRM 3264 St. John’s Wort

(Hypericum perforatum L.) Methanol Extract was determined at NIST using isotope dilution 

inductively coupled plasma mass spectrometry (ID-ICP-MS). The NIST-determined value and 

uncertainty for As are provided in the table below, on an as-received basis. The certified values

and uncertainties for Pb are provided in the table below, both on a dry-mass basis and on an asreceived basis accounting for moisture of the material (0.92 %).
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NIST-Determined Mass Fraction in SRM 3264 (ng/g)

Analyte (as-received basis)

Arsenic (As) 50 ± 18

Certified Mass Fraction in SRM 3264 (ng/g)

Analyte (dry-mass basis) (as-received basis)

Lead (Pb) 30.3 ± 1.8 30.0 ± 1.8

Study Results

• Fifty-nine laboratories enrolled in this exercise and received samples.

• Forty laboratories reported results for arsenic in St. John’s wort aerial parts (68 % 

participation). Forty-two laboratories reported results for lead in St. John’s wort aerial 

parts (71 % participation). Thirty-seven and 42 laboratories were used, respectively, 

for calculation purposes, see Statistics, page 3. 

• Thirty-seven laboratories reported results for arsenic in St. John’s wort methanol 

extract (63 % participation). Thirty-eight laboratories reported results for lead in St. 

John’s wort methanol extract (64 % participation). Thirty-four laboratories were used 

in both studies for calculation purposes, see Statistics, page 3.

• The consensus means for arsenic in the St. John’s wort aerial parts and methanol extract

were within the target ranges with high between-laboratory variability (23 % and 30 % 

RSD, respectively).

• The consensus mean for lead in the St. John’s wort aerial parts was within the target range 

with acceptable between-laboratory variability (13 % RSD). The consensus mean for lead 

in the St. John’s wort methanol extract was slightly above the target range with high

between-laboratory variability (28 % RSD).

• For arsenic, a majority of the laboratories reported using microwave digestion (81 %) for 

sample preparation. Hot block digestion (14 %) and open beaker digestion (5 %) were also 

reported as methods of sample preparation.

• For lead, a majority of the laboratories also reported using microwave digestion (79 %) for 

sample preparation. Hot block digestion (14 %) and open beaker digestion (7 %) were also 

reported as methods of sample preparation.

• For arsenic, most laboratories reported using ICP-MS as their analytical method for 

analysis (90 %). Laboratories also reported using AAS (5 %) and ICP-OES (5 %).

• For lead, most laboratories also reported using ICP-MS as their analytical method for 

analysis (88 %). Laboratories also reported using AAS (9 %) and ICP-OES (2 %).

Technical Recommendations

The following recommendations are based on results obtained from the participants in this study.

• Loss of volatile species of As is a concern and care must be taken not to lose As during 

sample preparation.

• With a vigorous microwave digestion (81 % reported using microwave sample 

preparation) the high temperatures should convert all volatile organoarsenic species to 

arsenate As(V). At this point any subsequent heating will not result in loss of arsenic.
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• Some laboratories performed well on the plant material but reported values with a high 

bias for the lower-level extract material.

• More accurate measurements can be achieved using a calibration curve which closely

surrounds the low concentrations found in these sample solutions.

• The concentrations of the sample solutions must lie within the linear section of the

calibration curve to prevent erroneous results. For a result outside the calibration range, 

multiplication by a dilution factor will only magnify the error.

• Some laboratories reported values within the target range for As in the extract material but 

reported low values in the plant material.

• Ensure that samples are completely digested; higher temperatures or a stronger acid 

such as HF may be needed for plant materials.

• Ensure that As is not lost during sample preparation either during inadvertent venting 

of vessels or when open beaker digestion is used.

• Lead is easily digested and volatile loss of Pb is not a concern. However, digestion with 

HCl may form a highly insoluble PbCl2 precipitate. Digestion with HNO3 is recommended

for Pb analysis, or dry ashing with a small volume of acid.

• ICP-MS or AAS are recommend for analysis of low levels of As and Pb. Sensitivity of As 

and Pb is poor when using ICP-OES, possible pre-concentration of sample solutions to 

overcome poor sensitivity may be of use but extra care should be taken to overcome any 

additional contamination issues.

• An appropriate number of procedural blanks are important, and can be critical when sample

concentrations are near the detection limit.
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Table 3. Individualized data summary table (NIST) for arsenic and lead in St. John’s wort (SJW) dietary supplements.

Lab Code: NIST

Analyte Sample Units xi si Z'comm ZNIST N x* s* xNIST U 95

Lead (Pb) SJW Aerial Parts ng/g 933 273 0.00 42 825 106 933 273

Lead (Pb) SJW Extract ng/g 30.0 3.6 0.00 34 34.0 9.0 30.0 3.6

Arsenic (As) SJW Aerial Parts ng/g 145 26 0.00 37 129 30 145 26

Arsenic (As) SJW Extract ng/g 49.6 36.0 0.00 34 41.9 12.4 49.6 36.0

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U95 ±95% confidence interval

Z'comm Z'-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

National Institute of Standards & Technology

Exercise L - October 2015 - Toxic Elements

1. Your Results 2. Community Results 3. Target
17

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Table 4. Data summary table for arsenic in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 145 26 49.6 36.0

L102 < 500.0 < 500.0 < 500.0 < 500.0 < 500.0 < 500.0

L103 123 154 139 22 < 60.0 < 60.0

L104

L105

L106 125 121 121 122 2 34.1 38.9 35.2 36.1 2.5

L107 178 182 187 182 5 40.9 42.2 40.9 41.3 0.8

L108 100 101 101 101 1 27.0 28.0 28.0 27.7 0.6

L109 113 108 107 110 3 31.8 31.6 32.5 32.0 0.5

L110 144 144 148 145 2 78.8 75.8 76.2 76.9 1.6

L111 123 112 105 113 9 55.0 45.0 54.0 51.3 5.5

L112

L115 119 129 126 125 5 32.0 31.0 31.0 31.3 0.6

L116 150 152 146 149 3 36.0 38.0 39.0 37.7 1.5

L117

L118 160 140 120 140 20 50.0 40.0 40.0 43.3 5.8

L120 108 109 108 108 1 32.6 36.4 33.4 34.1 2.0

L122

L123 168 155 147 157 11 39.9 42.2 48.5 43.5 4.5

L124

L125 164 156 160 6 40.0 38.4 36.9 38.4 1.6

L126 103 101 97 100 3 32.7 32.8 34.9 33.5 1.2

L127 < 5.0 < 5.0 < 5.0 6.5 11.9 < 5.0 9.2 3.8

L128 114 122 105 114 9 94.0 90.0 93.0 92.3 2.1

L129 160 160 170 163 6

L130

L131

L132

L134

L136 102 104 98 101 3 23.5 22.5 22.9 23.0 0.5

L137 98 100 99 99 1 43.0 45.0 42.7 43.6 1.3

L138 173 181 186 180 7 37.4 50.9 49.1 45.8 7.3

L139 80 80 70 77 6 37.3 30.6 40.5 36.1 5.1

L140 114 112 122 116 5 37.0 38.4 36.1 37.2 1.2

L141 119 114 121 118 4 38.2 35.6 37.4 37.1 1.3

L142 87 77 91 85 7

L143

L144 91 99 81 90 9 58.0 37.0 40.0 45.0 11.4

L145 142 143 147 144 3 43.7 44.4 41.5 43.2 1.5

L147 134 139 172 148 21 37.0 35.5 219.8 97.4 106.0

L148 141 148 147 145 4 62.3 70.5 67.3 66.7 4.1

L151 108 105 112 108 3 36.3 32.7 41.3 36.8 4.3

L154

L155 114 111 106 110 4 36.8 24.4 35.9 32.4 6.9

L156

L157 108 112 114 111 3 32.9 37.0 34.3 34.7 2.1

L158 138 141 141 140 2 49.0 57.0 48.0 51.3 4.9

L159

L160 140 130 151 140 11 36.4 40.3 40.2 39.0 2.2

L161

L162

L165 140 170 170 160 17 40.0 40.0 30.0 36.7 5.8

L166

L167 149 141 151 147 5 95.5 89.0 101.0 95.2 6.0

L169 162 157 157 159 3 69.8 71.3 67.1 69.4 2.1

L170 107 105 98 103 5 45.0 49.0 49.0 47.7 2.3

L171

L172

L173 215 220 220 218 3

L174

L175

L177

L179 < 330.0 < 330.0 < 330.0 < 520.0 < 520.0 < 520.0

L180

L181

 Consensus Mean 129 Consensus Mean 41.9

 Consensus Standard Deviation 30 Consensus Standard Deviation 12.4

 Maximum 218 Maximum 97.4

 Minimum 77 Minimum 9.2

 N 37 N 34

Total Arsenic

SRM 3262 St. John's Wort Aerial Parts (ng/g) SRM 3264 St. John's Wort Extract (ng/g)

Individual Results Community Results
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Table 5. Data summary table for lead in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 933 273 30.0 3.6

L102 899 834 885 873 34 < 200.0 < 200.0 < 200.0

L103 800 811 806 8 < 60.0 < 60.0

L104

L105

L106 889 760 879 843 72 28.8 28.3 27.3 28.1 0.8

L107 748 722 741 737 13 25.8 26.6 25.8 26.1 0.5

L108 697 693 693 694 2 32.0 32.0 32.0 32.0 0.0

L109 763 744 680 729 43 38.6 40.3 26.1 35.0 7.8

L110 819 851 849 840 18 42.0 41.4 43.0 42.1 0.8

L111 866 791 769 809 51 30.0 29.0 31.0 30.0 1.0

L112

L115 796 767 831 798 32 28.0 27.0 29.0 28.0 1.0

L116 862 884 887 878 14 32.0 35.0 32.0 33.0 1.7

L117

L118 730 810 870 803 70 30.0 30.0 30.0 30.0 0.0

L120 965 729 814 836 120 37.5 37.5 41.1 38.7 2.1

L122

L123 756 725 849 777 65 24.5 24.0 22.8 23.8 0.9

L124

L125 850 887 869 26 29.0 31.7 32.1 30.9 1.7

L126 757 771 634 721 75 38.5 35.8 38.5 37.6 1.6

L127 714 714 896 775 105 < 5.0 < 5.0 < 5.0

L128 997 946 999 981 30 35.0 32.0 33.0 33.3 1.5

L129 980 990 930 967 32

L130

L131

L132

L134

L136 683 637 640 653 26 49.5 37.0 41.3 42.6 6.4

L137 761 790 748 766 22 66.7 67.1 74.1 69.3 4.2

L138 986 952 904 947 41 59.6 81.2 41.2 60.7 20.0

L139 680 670 670 673 6 24.7 23.0 24.1 23.9 0.9

L140 755 721 860 779 72 26.9 28.8 31.1 28.9 2.1

L141 744 795 834 791 45 33.3 31.6 32.8 32.6 0.9

L142 815 922 968 902 79 45.0 45.0

L143

L144 800 898 898 865 57 118.0 126.0 115.0 119.7 5.7

L145

L147 807 868 852 842 32 25.6 24.2 26.5 25.4 1.2

L148 740 842 671 751 86 28.2 25.7 24.9 26.3 1.7

L151 698 715 745 719 24 44.2 50.5 43.8 46.2 3.8

L154 1000 988 1008 999 10

L155 813 756 744 771 37 27.6 22.9 25.9 25.5 2.4

L156

L157 689 779 845 771 78 27.2 29.1 37.0 31.1 5.2

L158 796 981 1170 982 187 32.0 32.0 31.0 31.7 0.6

L159

L160 756 823 909 829 77 28.9 28.5 28.3 28.6 0.3

L161

L162

L165 860 920 830 870 46 30.0 40.0 40.0 36.7 5.8

L166

L167 813 806 712 777 56 58.9 67.5 69.1 65.2 5.5

L169 921 947 947 938 15 45.1 45.6 46.9 45.9 0.9

L170 693 773 645 704 65 26.0 26.0 29.0 27.0 1.7

L171

L172

L173 772 778 775 775 3 20.3 20.5 21.0 20.6 0.4

L174

L175

L177

L179 940 810 860 870 66 25.0 29.0 28.0 27.3 2.1

L180 1050 1000 1010 1020 26

L181 1050 1000 1010 1020 26

 Consensus Mean 825 Consensus Mean 34.0

 Consensus Standard Deviation 106 Consensus Standard Deviation 9.0

 Maximum 1020 Maximum 119.7

 Minimum 653 Minimum 20.6

 N 42 N 34

Lead

SRM 3262 St. John's Wort Aerial Parts (ng/g) SRM 3264 St. John's Wort Extract (ng/g)

Community 

Results Individual Results
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Figure 4. Arsenic in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – digestion and analytical 

method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The 

color of the data point represents the sample preparation (digestion) procedure and analytical method employed. The black solid line 

represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and below 

the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” 

performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
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Figure 5. Arsenic in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – digestion and 

analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation (digestion) procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
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Figure 6. Lead in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – digestion and analytical 

method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The 

color of the data point represents the sample preparation (digestion) procedure and analytical method employed. The black solid line 

represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and below 

the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” 

performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
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Figure 7. Lead in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – digestion and 

analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation (digestion) procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST certified value bounded by twice its uncertainty (U95).
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Figure 8. Arsenic in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort (Hypericum 

perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one sample (St. 

John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The solid red box represents 

the target zone for the two samples, St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis). The dotted blue 

box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
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Figure 9. Lead in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort (Hypericum 

perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one sample (St. 

John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The solid red box represents 

the target zone for the two samples, St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis). The dotted blue 

box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
25

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WATER-SOLUBLE VITAMINS (BIOTIN) IN DIETARY SUPPLEMENTS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 3290 Dry Cat Food and 

SRM 3280 Multivitamin/Multielement Tablets. Participants were asked to use in-house analytical 

methods to determine the mass fraction of biotin in each of the matrices and report values on an 

as-received basis.

Sample Information

Cat Food. Participants were provided with one packet containing approximately 10 g of dry cat 

food. The cat food was blended, aliquotted, and heat-sealed inside 4 mil polyethylene bags, which 

were then sealed inside nitrogen-flushed aluminized plastic bags along with two packets of silica 

gel. Before use, participants were instructed to thoroughly mix the contents of each packet and to 

use a sample size of at least 0.5 g. Participants were asked to store the material at controlled room 

temperature, 10 °C to 30 °C, and to prepare three samples and report three values from the single 

packet provided. Approximate analyte levels were not reported to participants prior to the study. 

The certified value for biotin in SRM 3290 Dry Cat Food was determined at NIST using isotope 

dilution liquid chromatography mass spectrometry (ID-LC-MS), in combination with data from 

numerous collaborating laboratories. The certified values and uncertainties for biotin are provided 

in the table below, both on a dry-mass basis and on an as-received basis accounting for moisture 

of the material (4.36 %).

Certified Mass Fraction in SRM 3290 (mg/kg)

Analyte (dry-mass basis) (as-received basis)

Biotin 1.42 ± 0.23 1.36 ± 0.22

Multivitamin. Participants were provided with one bottle containing 30 multivitamin/multielement 

tablets. Before use, participants were instructed to grind all tablets together and mix the resulting 

powder thoroughly, and to use a sample size of at least 1.0 g. Participants were asked to store the 

material at controlled room temperature, 10 °C to 30 °C, and to prepare three samples and report 

three values from the single bottle provided. Approximate analyte levels were not reported to 

participants prior to the study. The certified value for biotin in SRM 3280 Multivitamin/

Multielement Tablets was determined at NIST using isotope dilution liquid chromatography mass 

spectrometry (ID-LC-MS), in combination with data from numerous collaborating laboratories. 

The certified values and uncertainties for biotin are provided in the table below, both on a drymass basis and on an as-received basis accounting for moisture of the material (1.37 %).

Certified Mass Fraction in SRM 3280 (mg/kg)

Analyte (dry-mass basis) (as-received basis)

Biotin 23.4 ± 3.2 23.1 ± 3.2

Study Results

• Forty laboratories enrolled in this exercise and received samples. Twenty-one laboratories 

reported results for SRM 3290 (53 % participation) and 23 laboratories reported results for 
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SRM 3280 (58 % participation). Nineteen and 23 laboratories were used, respectively, for 

calculation purposes, see Statistics, page 3.

• The consensus mean was within the target range for biotin in the multivitamin with 

acceptable between-laboratory variability (20 % RSD).

• The consensus mean was above the target range for biotin in the cat food with very high 

between-laboratory variability (61 % RSD).

• A majority of the laboratories reported using solvent extraction (78 %) as the sample 

preparation method. Laboratories also reported using dilution (13 %) and no sample 

preparation (9 %).

• A majority of the laboratories reported using liquid chromatography with mass 

spectrometry (48 %) as their instrumental method for analysis. Use of LC with absorbance 

detection (35 %), LC with tandem mass spectrometry (9%), HPLC (4 %), and 

microbiological assay (4 %) were also reported.

Technical Recommendations

The following recommendations are based on results obtained from the participants in this study.

• Results for the multivitamin tablet were excellent. No methods presented as significantly 

better or worse than any other. No systematic biases were noted.

• For the cat food matrix, laboratories utilizing highly specific tandem mass spectrometry 

methods reported the most accurate results compared to the target value. The results from 

the single laboratory reporting use of microbiological assay were also consistent with the 

target value.

• Several laboratories reported values in the target range for the multivitamin tablet but high 

results for the cat food, indicating a potential challenge with the cat food matrix.

• Many of the laboratories reporting near the consensus mean, but higher than the target 

range, reported using LC-MS based methods. The high bias could be a result of a coelution 

or ion enhancement/suppression effects if an appropriate internal standard is not utilized.

• Extreme outliers in the measurement of biotin are likely a result of lack of specificity in 

the instrumental method.

• All of the outlying laboratories reporting extremely high values used LC-absorbance

methods.

• Some laboratories using LC-absorbance may be experiencing a co-elution that would 

cause a high bias in the results. The problem can likely be corrected by alteration of 

the chromatographic conditions. The following recommendations can help identify 

and avoid potential coelutions.

• A chromatographic method with alternate selectivity (different retention order) can 

be used as a check for each new sample type that is run. Ideally, the retention of 

coeluting compounds would also be affected and the results from the two 

chromatographic systems would be different. Two different responses would 

indicate a possible bias in one approach.

• A different detector can be used in series with an absorbance detector (as 

confirmation), such as a fluorescence detector or mass spectrometer. If a coeluting 

compound is present, the response from these detectors would be different than the 

response from the absorbance detector. Two different responses would indicate a 

possible bias in one approach.
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• Considerations of potential interferences can assist in troubleshooting. 

Understanding the matrix that is being tested and possible coeluting compounds 

can be evaluated before a sample is analyzed for additional confidence in the result.
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Table 6. Individualized data summary table (NIST) for biotin in dietary supplements.

Lab Code: NIST

Analyte Sample Units xi si Z'comm ZNIST N x* s* xNIST U 95

Biotin Multivitamin mg/kg 23.1 6.3 0.00 23 23.9 4.9 23.1 6.3

Biotin Cat Food mg/kg 1.36 0.44 0.00 19 2.39 1.45 1.36 0.44

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U95 ±95% confidence interval

Z'comm Z'-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

National Institute of Standards & Technology

Exercise L - October 2015 - Biotin

1. Your Results 2. Community Results 3. Target
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Table 7. Data summary table for biotin in dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 23.1 6.3 1.36 0.44

L104

L105

L107 23.7 22.7 24.5 23.6 0.9 1.32 1.26 1.33 1.30 0.04

L108

L110 24.0 22.6 22.7 23.1 0.8 1.27 1.18 1.26 1.24 0.05

L111 20.6 20.6 20.8 20.7 0.1

L112

L116 21.6 22.1 21.4 21.7 0.4 1.50 1.70 1.70 1.63 0.12

L117

L118 21.2 21.1 21.4 21.2 0.2 1.23 1.28 1.21 1.24 0.04

L120 23.1 22.1 22.8 22.7 0.5 1.31 1.25 1.58 1.38 0.18

L121 25.40 24.20 24.90 24.83 0.60

L122

L123 30.3 33.1 34.3 32.6 2.1 3.66 4.00 4.03 3.90 0.21

L124

L125 23.7 23.1 23.4 0.4

L126 23.1 23.4 23.8 23.4 0.4 3.88 3.62 3.93 3.81 0.17

L127 71600.0 68667.0 68000.0 69422.3 1915.2 < 50.000 < 50.000 < 50.000

L128 40.9 41.8 41.5 41.4 0.5 112.00 112.00 111.00 111.67 0.58

L130

L134

L137 116.9 120.8 134.8 124.1 9.4 7.22 7.58 6.85 7.22 0.37

L138 22.0 22.0 22.0 22.0 0.0 1.60 1.60 1.30 1.50 0.17

L139 23.6 23.1 23.1 23.3 0.3 1.10 1.12 1.07 1.10 0.03

L140 23.2 23.1 23.2 23.2 0.1 1.83 1.74 1.66 1.74 0.09

L141 24.7 24.6 25.5 24.9 0.5 2.08 2.38 2.12 2.19 0.16

L142

L148

L151 22.6 22.7 23.1 22.8 0.3 2.91 2.98 2.92 2.93 0.04

L153

L155 22.5 24.1 26.2 24.3 1.9 1.76 1.90 2.03 1.90 0.14

L157 20.8 21.6 21.1 21.1 0.4 3.00 2.74 2.85 2.86 0.13

L158 15.5 14.1 14.3 14.6 0.8 1.41 1.38 1.45 1.41 0.04

L159

L160

L166

L168 20.2 20.8 21.4 20.8 0.6 1.26 1.39 1.40 1.35 0.08

L170 30.6 29.9 29.1 29.9 0.8

L171

L172

L177

L178

L179 10.9 11.2 12.1 11.4 0.6 < 0.380 < 0.380 < 0.380

 Consensus Mean 23.9 Consensus Mean 2.39

 Consensus Standard Deviation 4.9 Consensus Standard Deviation 1.45

 Maximum 69422.3 Maximum 111.67

 Minimum 11.4 Minimum 1.10

 N 23 N 19

Individual Results

SRM 3280 MultivitaminTablets (mg/kg) SRM 3290 Dry Cat Food (mg/kg)

Biotin

Community 

Results
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Figure 10. Biotin in SRM 3290 Dry Cat Food (data summary view – sample preparation and analytical method). In this view, individual 

laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The color of the data point represents 

the sample preparation procedure and analytical method employed. The black solid line represents the consensus mean, and the green

shaded region represents the range of tolerance, calculated as the values above and below the consensus mean that result in an acceptable 

Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, which encompasses the NIST 

certified value bounded by twice its uncertainty (U95).
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Figure 11. Biotin in SRM 3280 Multivitamin/Multielement Tablets (data summary view – sample preparation and analytical method). 

In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The color of 

the data point represents the sample preparation procedure and analytical method employed. The black solid line represents the 

consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and below the consensus 

mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, 

which encompasses the NIST certified value bounded by twice its uncertainty (U95).
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Figure 12. Biotin in SRM 3290 Dry Cat Food and SRM 3280 Multivitamin/Multielement Tablets (sample/sample comparison view). 

In this view, the individual laboratory results for one sample (cat food) are compared to the results for a second sample (multivitamin). 

The solid red box represents the target zone for the two samples, cat food (x-axis) and multivitamin (y-axis). The dotted blue box 

represents the consensus zone for cat food (x-axis) and multivitamin (y-axis).
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XANTHOPHYLLS (LUTEIN AND ZEAXANTHIN) IN DIETARY SUPPLEMENTS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 2385 Slurried Spinach and 

SRM 3280 Multivitamin/ Multielement Tablets. Participants were asked to use in-house analytical 

methods to determine the mass fractions of lutein and zeaxanthin in each of the matrices and report 

values on an as-received basis.

Sample Information

Spinach. Participants were provided with one jar containing approximately 70 g of slurried 

spinach. The pureed spinach was blended, aliquotted, and sealed inside 2.5-oz. jars. Before use, 

participants were instructed to mix the contents of the jar thoroughly, and use a sample size of at 

least 1.5 g. Participants were asked to store the material under refrigeration, 0 °C to 4 °C, and to 

prepare three samples and report three values from the single jar provided. Approximate analyte 

levels were not reported to participants prior to the study. The certified value and uncertainty for 

total lutein in SRM 2385 was determined at NIST by LC-absorbance following solvent extraction

with and without saponification, in combination with data from numerous collaborating 

laboratories. The certified value and uncertainty are reported in the table below on an as-received

basis. The target value and uncertainty for zeaxanthin in SRM 2385 was determined at NIST by 

LC-absorbance following solvent extraction without saponification. The NIST-determined value 

and uncertainty are reported in the table below on an as-received basis.

Certified Mass Fraction in SRM 2385 (mg/kg)

Analyte (as-received basis)

Total Lutein 32.9 ± 6.5

NIST-Determined Mass Fraction in SRM 2385 (mg/kg)

Analyte (as-received basis)

Free Zeaxanthin 0.450 ± 0.080

Multivitamin/Multielement Tablets. Participants were provided with one bottle containing 30 

multivitamin/multielement tablets. Before use, participants were instructed to grind all 30 tablets, 

mix the resulting powder thoroughly, and use a sample size of at least 2.0 g. Participants were 

asked to store the material at controlled room temperature, 10 °C to 30 °C, prepare three samples, 

and report three values from the single bottle provided. Approximate analyte levels were not 

reported to participants prior to the study. The certified value and uncertainty for lutein in 

SRM 3280 was determined by LC-absorbance following solvent extraction, in combination with 

data from numerous collaborating laboratories. The certified value and uncertainty are reported 

in the table below on a dry-mass basis and after correction for moisture of the material (1.37 %). 

The target value and uncertainty for zeaxanthin in SRM 3280 was determined at NIST by LCabsorbance following solvent extraction without saponification. The NIST-determined value and 

uncertainty are reported in the table below on an as-received basis.
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Certified Mass Fraction in SRM 3280 (mg/kg)

Analyte (dry-mass basis) (as received basis)

Total Lutein 205 ± 50 202 ± 49

NIST-Determined Mass Fraction in SRM 3280 (mg/kg)

Analyte (as-received basis)

Total Zeaxanthin 5.4 ± 0.5

Study Results

• Forty laboratories enrolled in this exercise and received samples.

• Thirteen laboratories reported results for lutein in the spinach sample (33 % 

participation). Seven laboratories reported results for zeaxanthin in the spinach sample 

(18 % participation). Thirteen and five laboratories were used, respectively, for 

calculation purposes, see Statistics, page 3.

• Sixteen laboratories reported results for lutein in the multivitamin (40 % participation). 

Twelve laboratories reported results for zeaxanthin in the multivitamin (30 % 

participation). Sixteen and 12 laboratories were used, respectively, for calculation 

purposes, see Statistics, page 3.

• The consensus mean for lutein in the spinach was near the bottom of the target range with 

high between-laboratory variability (37 % RSD). The consensus mean for lutein in the 

multivitamin was within the target range with acceptable between-laboratory variability

(15 % RSD).

• The consensus mean for zeaxanthin in the spinach was above the target range with 

extremely high between-laboratory variability (>100 % RSD). The consensus mean for 

zeaxanthin in the multivitamin was above the target range with high between-laboratory 

variability (30 % RSD).

• A majority of the laboratories reported using solvent extraction (86 %) as the sample 

preparation method. Some laboratories also reported using saponification (7 %), dilution 

(7 %), or no sample preparation technique (7 %).

• A majority of the laboratories reported using LC-absorbance (87 %) as their instrumental 

method for analysis. HPLC (7 %) and LC with a Diode Array Detector (LC-DAD, 7 %) 

were also reported by some laboratories.

Technical Recommendations

The following recommendations are based on results obtained from the participants in this study. 

• Care should be taken to minimize losses during the extraction process, during solvent 

evaporation, and by carefully washing down container walls with several rinses during 

each step to ensure complete dissolution of any residues.

• In general, laboratories reporting more vigorous extraction procedures, i.e. those using 

hexanes and longer extraction times, reported results closer to the target value.

• Since loss by photodecomposition is possible, care should be taken to prevent such losses 

(use of amber vials, aluminum foil, and/or reduced lighting).

• When using LC-absorbance, chromatographic coelutions may cause results to be biased 

high. This is particularly important if monitoring the absorbance in the UV where many 
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other compounds may also have chromophores. To avoid a high bias, more selective 

detectors (fluorescence, mass spectrometry) or chromatography with alternate selectivity

may be used.

• When making calibration solutions make sure they are of known quality. These may need 

to be tested before running samples, which may include determination of purity by 

chromatographic and spectroscopic methods.

• If using an internal standard, the internal standard must behave similarly to the analyte of 

interest in extraction, chromatographic analysis, and detection.
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Table 8. Individualized data summary table (NIST) for lutein and zeaxanthin in dietary supplements.

Lab Code: NIST

Analyte Sample Units xi si Z'comm ZNIST N x* s* xNIST U 95

Lutein Multivitamin mg/kg 202 99 0.00 16 175 27 202 99

Lutein Spinach mg/kg 32.9 13.0 0.00 13 21.5 8.0 32.9 13.0

Zeaxanthin Multivitamin mg/kg 5.40 0.52 0.00 12 11.09 3.30 5.40 0.52

Zeaxanthin Spinach mg/kg 0.450 0.160 0.00 5 1.591 1.746 0.450 0.160

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U95 ±95% confidence interval

Z'comm Z'-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

National Institute of Standards & Technology

Exercise L - October 2015 - Xanthophylls

1. Your Results 2. Community Results 3. Target
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Table 9. Data summary table for lutein in dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 202 99 32.9 13.0

L101

L102

L103

L104

L105

L107 166 164 165 165 1 18.6 18.8 18.6 18.7 0.1

L110 167 171 160 166 6

L111 108 104 111 108 4

L112

L113 180 181 169 177 7 24.9 24.2 24.2 24.4 0.4

L116

L117

L118 200 186 187 191 8 25.2 24.2 25.6 25.0 0.7

L119 152 148 141 147 5 7.2 7.2 7.4 7.3 0.1

L121 144 177 155 159 17 20.5 19.7 19.8 20.0 0.5

L122

L123 269 251 263 261 9 8.6 9.8 8.7 9.0 0.7

L128 174 154 175 168 12 21.0 21.0 21.0 21.0 0.0

L130

L134

L137 166 156 173 165 8 19.3 19.5 24.1 21.0 2.7

L138

L139 164 178 159 167 10 20.8 20.8 22.6 21.4 1.0

L144

L145

L150

L158

L159

L166

L167 225 211 221 219 7 30.3 33.0 29.8 31.0 1.7

L168 162 174 169 168 6 28.1 26.1 26.2 26.8 1.2

L170 182 181 181 181 1

L171 975 1015 1035 1008 31 43.0 44.0 42.0 43.0 1.0

L172

L177

L179 173 185 161 173 12 17.8 17.9 17.7 17.8 0.1

 Consensus Mean 175 Consensus Mean 21.5

 Consensus Standard Deviation 27 Consensus Standard Deviation 8.0

 Maximum 1008 Maximum 43.0

 Minimum 108 Minimum 7.3

 N 16 N 13

Community 

Results

Lutein

SRM 3280 MultivitaminTablets (mg/kg) SRM 2385 Slurried Spinach (mg/kg)

Individual Results
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Table 10. Data summary table for zeaxanthin in dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 5.4 0.5 0.450 0.160

L101

L102

L103

L104

L105

L107 13.4 13.7 13.7 13.6 0.2 0.638 0.646 0.644 0.643 0.004

L110 13.0 13.4 12.5 13.0 0.5

L111 7.5 7.4 7.8 7.6 0.2

L112

L113 12.5 12.6 11.5 12.2 0.6 3.053 2.898 3.013 2.988 0.080

L116

L117

L118 12.1 11.2 11.0 11.4 0.6 < 1.000 < 1.000 < 1.000

L121 8.9 8.3 10.1 9.1 0.9 0.459 0.397 0.453 0.436 0.034

L122

L128 13.0 10.0 13.0 12.0 1.7

L130

L137 7.1 6.9 8.8 7.6 1.1

L138

L139 13.5 12.0 12.0 12.5 0.9 < 1.000 < 1.000 < 1.000

L144

L150

L158

L159

L160

L166

L167 31.4 29.6 33.4 31.5 1.9 3.900 3.400 3.300 3.533 0.321

L168

L170 11.9 12.3 11.6 11.9 0.4

L172

L177

L179 4.7 4.7 4.6 4.7 0.1 0.350 0.360 0.360 0.357 0.006

 Consensus Mean 11.1 Consensus Mean 1.591

 Consensus Standard Deviation 3.3 Consensus Standard Deviation 1.746

 Maximum 31.5 Maximum 3.533

 Minimum 4.7 Minimum 0.357

 N 12 N 5

Zeaxanthin

SRM 3280 MultivitaminTablets (mg/kg) SRM 2385 Slurried Spinach (mg/kg)

Community 

Results Individual Results
39

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Figure 13. Lutein in SRM 2385 Slurried Spinach (data summary view – sample preparation and analytical method). In this view, 

individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The color of the data point 

represents the sample preparation procedure and analytical method employed. The black solid line represents the consensus mean, and 

the green shaded region represents the range of tolerance, calculated as the values above and below the consensus mean that result in an 

acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, which encompasses the 

NIST certified value bounded by twice its uncertainty (U95).
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Figure 14. Lutein in SRM 3280 Multivitamin/Multielement Tablets (data summary view – sample preparation and analytical method). 

In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The color of 

the data point represents the sample preparation procedure and analytical method employed. The black solid line represents the 

consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and below the consensus 

mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, 

which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
41

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Figure 15. Zeaxanthin in SRM 2385 Slurried Spinach (data summary view – sample preparation and analytical method). In this view, 

individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The color of the data point 

represents the sample preparation procedure and analytical method employed. The black solid line represents the consensus mean, and 

the green shaded region represents the range of tolerance, calculated as the values above and below the consensus mean that result in an 

acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, which encompasses the 

NIST certified value bounded by twice its uncertainty (U95).
42

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Figure 16. Zeaxanthin in SRM 3280 Multivitamin/Multielement Tablets (data summary view – sample preparation and analytical 

method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The 

color of the data point represents the sample preparation procedure and analytical method employed. The black solid line represents the 

consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and below the consensus 

mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, 

which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
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Figure 17. Lutein in SRM 2385 Slurried Spinach and SRM 3280 Multivitamin/Multielement Tablets (sample/sample comparison 

view). In this view, the individual laboratory results for one sample (spinach) are compared to the results for a second sample 

(multivitamin). The solid red box represents the target zone for the two samples, spinach (x-axis) and multivitamin (y-axis). The 

dotted blue box represents the consensus zone for spinach (x-axis) and multivitamin (y-axis).
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Figure 18. Zeaxanthin in SRM 2385 Slurried Spinach and SRM 3280 Multivitamin/Multielement Tablets (sample/sample 

comparison view). In this view, the individual laboratory results for one sample (spinach) are compared to the results for a second 

sample (multivitamin). The solid red box represents the target zone for the two samples, spinach (x-axis) and multivitamin (y-axis). 

The dotted blue box represents the consensus zone for spinach (x-axis) and multivitamin (y-axis).
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FATTY ACIDS IN FISH OILS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 3275-I Omega-3 and Omega6 Fatty Acids in Fish Oil and SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil. 

Participants were asked to use in-house analytical methods to determine the mass fractions of six 

fatty acids (linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, EPA, and DHA) in 

each of the matrices and report values on an as-received basis as fatty acid methyl esters (FAMEs).

Sample Information

Fish Oil 1. Participants were provided with three ampoules containing 1.2 mL of fish oil

concentrate high in DHA. The fish oil was combined with mixed natural tocopherols (minimum 

1 mg/g) as an antioxidant and ampouled under argon into 2 mL amber ampoules. Before use, 

participants were instructed to mix the contents of each ampoule thoroughly and use a sample size 

of at least 0.5 g. Participants were asked to store the material under refrigeration, 0 °C to 4 °C, 

and report a single value from each ampoule provided. Approximate analyte levels were not 

reported to participants prior to the study. The certified and reference values and uncertainties for 

fatty acids in SRM 3275-I were determined at NIST by gas chromatography with mass 

spectrometric detection (GC-MS) and GC with flame ionization detection (GC-FID). The certified 

and reference values and uncertainties are reported in the table below on an as-received basis.

Analyte

Certified and Reference Mass Fraction

in SRM 3275-I (mg/g as FAME)

Linoleic Acid 2.31 ± 0.19

α-Linolenic Acid 1.21 ± 0.05

γ-Linolenic Acid 0.344 ± 0.025

Arachidonic Acid 5.69 ± 0.19

EPA 113 ± 12

DHA 429 ± 15

Fish Oil 2. Participants were provided with three ampoules containing 1.2 mL of fish oil

concentrate containing 60 % long-chain omega-3 fatty acids. The fish oil was combined with 

mixed natural tocopherols (minimum 1 mg/g) as an antioxidant and ampouled under argon into 

2 mL amber ampoules. Before use, participants were instructed to mix the contents of each 

ampoule thoroughly and use a sample size of at least 0.5 g. Participants were asked to store the 

material under refrigeration, 0 °C to 4 °C, and report a single value from each ampoule provided. 

Approximate analyte levels were not reported to participants prior to the study. The certified and 

reference values and uncertainties for fatty acids in SRM 3275-I were determined at NIST by gas 

chromatography with mass spectrometric detection (GC-MS) and GC with flame ionization 

detection (GC-FID). The certified and reference values and uncertainties are reported in the table 

below on an as-received basis.
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Analyte

Certified and Reference Mass Fraction 

in SRM 3275-III (mg/g as FAME)

Linoleic Acid 13.49 ± 0.45

α-Linolenic Acid 6.61 ± 0.31

γ-Linolenic Acid 1.771 ± 0.099

Arachidonic Acid not assigned

EPA 154 ± 9

DHA 104 ± 5

Study Results

• Forty laboratories enrolled in this exercise and received samples. Seventeen to twentythree laboratories reported results (43 % to 58 % participation), depending on the analyte 

and matrix combination.

• In the first fish oil sample (SRM 3275-I, a concentrate high in DHA), the consensus means

for all fatty acids were within the target ranges.

• While within the target ranges, the consensus means for γ-linolenic acid, arachidonic 

acid, and DHA were near the upper bounds of the respective target ranges.

• The between-laboratory variability for EPA and DHA was excellent at 10 % and 14 % 

RSD, respectively.

• The variabilities for linoleic acid, α-linolenic acid, and arachidonic acid were 

acceptable at 25 % to 31 % RSD.

• Results for γ-linolenic acid displayed very high between-laboratory variability (76 % 

RSD).

• In the second fish oil sample (SRM 3275-III, a concentrate containing 60 % long-chain 

omega-3 fatty acids), only the consensus means for α-linolenic acid, EPA, and DHA were 

within the target ranges.

• While within the target ranges, the consensus means for α-linolenic acid and EPA were 

near the lower bounds of the respective target ranges. The consensus mean for DHA 

was near the upper bounds of the target range.

• The consensus mean for linoleic acid was below the target range.

• The consensus mean for γ-linolenic acid was above the target range.

• No target range was provided for arachidonic acid.

• The between-laboratory variability for EPA and DHA was acceptable at 22 % and 28 % 

RSD, respectively.

• The variabilities for linoleic acid, α-linolenic acid, γ-linolenic acid, and arachidonic 

acid were high at 32 % to 57 % RSD.

• A majority of laboratories reported using saponification or base hydrolysis (41 %) or 

derivatization (36 %) for sample preparation. Other reported techniques included acid 

hydrolysis (9 %), solvent extraction (9 %), and dilution (5 %).

• A majority of laboratories reported using gas chromatography with flame ionization 

detection (GC-FID) as their analytical method of analysis (91 %). GC with mass 

spectrometric detection (GC-MS) was also reported (9 %).
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Technical Recommendations

The following recommendations are based on results obtained from the participants in this study. 

• With a small number of laboratories reporting data for these fatty acids, and a majority 

reporting use of the same or very similar methods, drawing extensive technical 

conclusions is difficult.

• Participants were asked to report concentrations for fatty acids as fatty acid methyl esters 

(FAMEs). In this case, FAMEs should be used as calibrants or non-esterified fatty acids 

should be carried through the entire sample preparation procedure (hydrolysis and 

derivatization) to improve quantitation.

• Knowledge of calibrant response when carried through the derivatization procedure is 

necessary. For example, at NIST, calibrants for EPA and DPA give response factors of 

1.3 and 1.6, respectively, corresponding to 30 % or 60 % low bias in the quantitation of 

these compounds if not considered.

• Similarly, for those laboratories using GC-MS, quantitation for some compounds may be 

inaccurate as a result of non-unity response factors from EI fragmentation.
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Table 11. Individualized data summary table (NIST) for fatty acids in fish oils.

Lab Code: NIST

Analyte Sample Units xi si Z'comm ZNIST N x* s* xNIST U 95

Linoleic Acid Fish Oil 1 mg/g 2.31 0.38 0.00 20 2.24 0.57 2.31 0.38

Linoleic Acid Fish Oil 2 mg/g 13.49 0.90 0.00 20 11.28 3.62 13.49 0.90

α-Linolenic Acid Fish Oil 1 mg/g 1.21 0.10 0.00 19 1.26 0.35 1.21 0.10

α-Linolenic Acid Fish Oil 2 mg/g 6.61 0.62 0.00 20 6.31 2.31 6.61 0.62

γ-Linolenic Acid Fish Oil 1 mg/g 0.344 0.050 0.00 14 0.389 0.297 0.344 0.050

γ-Linolenic Acid Fish Oil 2 mg/g 1.77 0.20 0.00 18 2.12 1.21 1.77 0.20

Arachidonic Acid Fish Oil 1 mg/g 5.69 0.38 0.00 18 6.02 1.85 5.69 0.38

Arachidonic Acid Fish Oil 2 mg/g 18 11.0 4.4

EPA Fish Oil 1 mg/g 113 24 0.00 22 109 11 113 24

EPA Fish Oil 2 mg/g 154 18 0.00 21 145 32 154 18

DHA Fish Oil 1 mg/g 429 30 0.00 22 448 63 429 30

DHA Fish Oil 2 mg/g 104 10 0.00 21 109 31 104 10

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U95 ±95% confidence interval

Z'comm Z'-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

National Institute of Standards & Technology

Exercise L - October 2015 - Fatty Acids

1. Your Results 2. Community Results 3. Target
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Table 12. Data summary table for linoleic acid in fish oils.

A B C Avg SD A B C Avg SD

2.31 0.38 13.49 0.90

2.37 2.33 2.30 2.33 0.04 12.20 12.20 12.20 12.20 0.00

2.43 2.55 < 1.00 2.49 0.08 2.43 2.50 2.72 2.55 0.15

2.91 2.89 2.87 2.89 0.02 16.10 16.20 15.95 16.08 0.13

2.14 2.16 2.15 2.15 0.01 11.85 11.87 11.87 11.86 0.01

3.76 3.60 3.68 3.68 0.08 18.53 18.09 18.31 18.31 0.22

11.80 11.80 11.90 11.83 0.06

2.07 2.09 2.00 2.05 0.05

1.10 0.93 0.96 1.00 0.09 9.14 9.37 9.40 9.30 0.14

2.04 2.08 2.08 2.07 0.02 11.75 11.50 11.61 11.62 0.13

3.00 3.00 3.00 3.00 0.00 15.00 15.00 14.00 14.67 0.58

0.99 0.56 0.63 0.73 0.23 4.37 4.29 3.40 4.02 0.54

2.15 2.14 2.23 2.17 0.05 10.11 10.37 10.47 10.32 0.19

1.50 1.50 1.40 1.47 0.06 10.60 10.60 10.60 10.60 0.00

2.19 2.10 2.10 2.13 0.05 12.03 12.14 12.24 12.14 0.11

3.07 3.57 3.63 3.42 0.31 13.50 13.90 14.00 13.80 0.26

2.29 2.29 2.57 2.38 0.16 2.37 2.34 2.62 2.44 0.15

2.20 2.20 2.20 0.00 11.90 12.00 11.95 0.07

2.11 2.14 2.12 2.12 0.02 11.90 12.00 11.90 11.93 0.06

2.09 2.12 2.15 2.12 0.03 13.98 14.05 14.09 14.04 0.06

2.00 2.10 2.04 2.05 0.05 11.87 11.83 11.97 11.89 0.07

2.20 2.20 2.20 2.20 0.00 6.90 6.30 7.90 7.03 0.81

 Consensus Mean 2.24 Consensus Mean 11.28

 Consensus Standard Deviation 0.57 Consensus Standard Deviation 3.62

 Maximum 3.68 Maximum 18.31

 Minimum 0.73 Minimum 2.44

 N 20 N 20

Linoleic Acid

SRM 3275-I Fish Oil (mg/g) SRM 3275-III Fish Oil (mg/g)
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Table 13. Data summary table for α-linolenic acid in fish oils.

Lab A B C Avg SD A B C Avg SD

NIST 1.21 0.10 6.61 0.62

L103

L104

L105

L107 1.25 1.28 1.27 1.27 0.02 7.02 6.97 6.95 6.98 0.04

L110

L111 < 1.00 < 1.00 < 1.00 1.89 1.60 < 1.00 1.75 0.21

L112

L113 1.71 1.70 1.69 1.70 0.01 9.25 9.30 9.14 9.23 0.08

L114

L116 1.20 1.21 1.20 1.20 0.01 6.68 6.68 6.69 6.68 0.01

L117

L121 1.10 1.10 1.30 1.17 0.12 7.00 6.90 7.00 6.97 0.06

L125 1.21 1.23 1.19 1.21 0.02

L129

L130

L131 0.21 0.20 0.22 0.21 0.01 3.98 3.96 4.51 4.15 0.31

L133

L134

L135 1.15 0.80 1.23 1.06 0.23 7.06 6.99 7.03 7.03 0.04

L136 2.00 2.00 2.00 2.00 0.00 9.00 9.30 9.00 9.10 0.17

L137 2.40 2.40 1.78 2.19 0.36

L139 1.24 1.24 1.35 1.28 0.06 6.40 6.79 6.64 6.61 0.20

L140

L144

L146 0.60 0.60 0.70 0.63 0.06 5.90 6.00 5.90 5.93 0.06

L149 1.16 1.13 1.16 1.15 0.02 6.45 6.64 6.38 6.49 0.13

L151

L152 1.66 1.71 1.75 1.71 0.05 7.40 7.55 7.72 7.56 0.16

L155

L157

L158 1.33 1.33 1.29 1.32 0.02 1.28 1.20 1.21 1.23 0.04

L159

L160 1.40 1.40 1.40 0.00 7.00 7.00 7.00 0.00

L164

L165 1.75 1.79 1.76 1.77 0.02 10.60 10.80 10.80 10.73 0.12

L168 1.13 1.21 1.16 1.17 0.04 7.02 7.15 7.09 7.09 0.07

L170 0.90 0.97 0.94 0.94 0.04 6.22 6.15 6.24 6.20 0.05

L172

L176

L177

L179 1.20 1.10 1.20 1.17 0.06 4.00 3.60 4.50 4.03 0.45

L182 1.21 1.20 1.27 1.23 0.04 6.72 6.84 6.74 6.77 0.06

 Consensus Mean 1.26 Consensus Mean 6.31

 Consensus Standard Deviation 0.35 Consensus Standard Deviation 2.31

 Maximum 2.00 Maximum 10.73

 Minimum 0.21 Minimum 1.23

 N 19 N 20

Individual Results Community Results

SRM 3275-I Fish Oil (mg/g) SRM 3275-III Fish Oil (mg/g)

α-Linolenic Acid
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Table 14. Data summary table for γ-linolenic acid in fish oils.

Lab A B C Avg SD A B C Avg SD

NIST 0.344 0.050 1.77 0.20

L103

L104

L105

L107 < 0.550 < 0.550 < 0.550 1.79 1.99 1.95 1.91 0.11

L110

L111 < 1.000 < 1.000 < 1.000 6.32 < 1.000 < 1.000 6.32

L112

L113 0.466 0.446 0.457 0.456 0.010 2.87 2.89 2.84 2.87 0.03

L114

L116 0.290 0.260 0.270 0.273 0.015 1.90 1.90 1.88 1.89 0.01

L117

L121 2.40 2.50 2.40 2.43 0.06

L125 0.242 0.265 0.251 0.253 0.012

L129

L130

L131 0.060 0.059 0.060 0.060 0.001 3.09 3.10 3.34 3.18 0.14

L133

L134

L135 0.001 0.001 0.001 0.001 0.000 1.64 1.68 1.53 1.62 0.08

L136 13.000 13.000 13.000 13.000 0.000 11.00 9.00 4.00 8.00 3.61

L137 0.29 0.44 0.29 0.34 0.09

L139 0.110 0.120 0.110 0.113 0.006 0.33 0.37 0.33 0.34 0.02

L140

L144

L146 2.900 2.700 2.800 2.800 0.100 3.10 3.20 3.20 3.17 0.06

L149 0.275 0.367 0.308 0.317 0.047 1.79 1.84 1.84 1.82 0.03

L151

L152 0.470 0.620 0.610 0.567 0.084 2.44 2.35 2.35 2.38 0.05

L155

L157

L158 0.460 0.580 0.960 0.667 0.261 0.40 0.68 0.66 0.58 0.16

L159

L160 0.370 0.350 0.360 0.014 2.00 2.00 2.00 0.00

L164

L165 < 0.100 < 0.100 < 0.100 2.46 2.48 2.45 2.46 0.02

L168 0.390 0.420 0.430 0.413 0.021 1.82 1.93 1.89 1.88 0.06

L170

L172

L176

L177

L179 0.300 0.300 0.300 0.300 0.000 1.30 1.20 1.50 1.33 0.15

L182

 Consensus Mean 0.389 Consensus Mean 2.12

 Consensus Standard Deviation 0.297 Consensus Standard Deviation 1.21

 Maximum 13.000 Maximum 8.00

 Minimum 0.001 Minimum 0.34

 N 14 N 18

Individual Results

γ-Linolenic Acid

SRM 3275-I Fish Oil (mg/g) SRM 3275-III Fish Oil (mg/g)

Community 

Results
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Table 15. Data summary table for arachidonic acid in fish oils.

Lab A B C Avg SD A B C Avg SD

NIST 5.69 0.38

L103

L104

L105

L107 5.94 5.89 5.87 5.90 0.04 13.20 13.10 13.20 13.17 0.06

L110

L111 7.26 6.68 7.01 6.98 0.29 6.30 6.93 6.69 6.64 0.32

L112

L113 8.32 8.35 8.28 8.31 0.04 17.33 17.45 17.19 17.32 0.13

L114

L116 6.07 6.10 6.14 6.10 0.04 12.89 12.85 12.81 12.85 0.04

L117

L119 5.55 5.58 5.56 5.56 0.02 12.88 12.73 12.80 12.80 0.08

L121 13.10 13.00 13.00 13.03 0.06

L125 11.80 12.00 11.70 11.83 0.15

L129

L130

L131 3.24 3.19 2.88 3.10 0.20 7.82 8.36 9.17 8.45 0.68

L134

L135 5.33 5.42 5.49 5.41 0.08 12.49 12.31 12.49 12.43 0.10

L136 1.00 1.00 1.00 1.00 0.00 3.00 11.00 10.00 8.00 4.36

L137 2.32 1.97 1.95 2.08 0.21 4.58 4.73 3.57 4.29 0.63

L139 6.33 6.20 6.08 6.20 0.13 14.72 15.06 15.42 15.07 0.35

L140

L144

L146 7.00 7.10 7.00 7.03 0.06 13.90 13.80 13.80 13.83 0.06

L149 6.97 6.68 7.05 6.90 0.19 13.23 13.09 13.47 13.26 0.19

L151

L152 7.84 8.18 8.24 8.09 0.22 14.80 15.30 15.30 15.13 0.29

L155

L157

L158 7.00 6.74 6.65 6.80 0.18 6.96 6.75 6.59 6.77 0.19

L159

L164

L165

L168 5.02 5.20 5.16 5.13 0.09 0.98 0.89 0.93 0.93 0.05

L170 5.59 5.77 5.65 5.67 0.09 13.04 12.87 13.08 13.00 0.11

L172

L176

L177

L179 5.90 5.70 5.70 5.77 0.12 7.70 7.00 8.80 7.83 0.91

L182

 Consensus Mean 6.02 Consensus Mean 11.02

 Consensus Standard Deviation 1.85 Consensus Standard Deviation 4.42

 Maximum 11.83 Maximum 17.32

 Minimum 1.00 Minimum 0.93

 N 18 N 18

Individual Results

Arachidonic Acid

SRM 3275-I Fish Oil (mg/g) SRM 3275-III Fish Oil (mg/g)

Community 

Results
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Table 16. Data summary table for EPA in fish oils.

Lab A B C Avg SD A B C Avg SD

NIST 113 24 154 18

L103

L104

L105

L107 107 107 108 107 1 155 155 154 155 1

L110

L111 110 118 112 113 4 111 113 114 113 2

L112

L113 142 144 142 143 1 206 207 205 206 1

L114

L116 112 112 112 112 0 157 158 158 158 1

L117

L119 110 110 110 110 0 157 156 157 157 1

L121 112 110 109 110 2 153 152 151 152 1

L125 106 107 104 106 2

L129

L130

L131 82 73 72 76 6 108 107 118 111 6

L133 114 115 115 1 111 112 112 1

L134

L135 99 102 104 102 3 148 149 147 148 1

L136 146 145 146 146 1 206 214 201 207 7

L137 45 37 37 40 5 58 59 47 55 7

L139 113 106 114 111 4 169 167 172 169 3

L140

L144

L146 103 103 101 102 1 146 146 145 146 1

L149 100 100 100 100 0 146 147 148 147 1

L151

L152 149 146 146 147 2 172 175 176 174 2

L155

L157

L158 106 106 106 106 0 106 106 106 106 0

L159

L160 92 95 94 94 2 136 137 135 136 1

L164

L165 110 111 109 110 1 159 160 160 160 1

L168 120 125 121 122 3 161 164 163 163 2

L170 104 108 106 106 2 158 156 158 157 1

L171

L172

L176

L177

L179 110 107 107 95 87 107

L182 107 107 109 108 1 153 155 153 154 1

 Consensus Mean 109 Consensus Mean 145

 Consensus Standard Deviation 11 Consensus Standard Deviation 32

 Maximum 147 Maximum 207

 Minimum 40 Minimum 55

 N 22 N 21

Individual Results

EPA

SRM 3275-I Fish Oil (mg/g) SRM 3275-III Fish Oil (mg/g)

Community 

Results
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Table 17. Data summary table for DHA in fish oils.

Lab A B C Avg SD A B C Avg SD

NIST 429 30 104 10

L103

L104

L105

L107 428 427 430 428 2 102 102 102 102 0

L110

L111 438 474 450 454 18 441 443 452 445 6

L112

L113 569 574 568 570 3 136 137 135 136 1

L114

L116 500 502 500 501 1 108 108 109 108 1

L117

L119 427 430 429 429 2 103 101 102 102 1

L121 447 444 438 443 5 101 100 99 100 1

L125 411 419 415 415 4

L129

L130

L131 265 232 243 247 17 55 54 60 56 3

L133 525 528 527 2 492 493 493 1

L134

L135 394 401 411 402 9 97 96 97 97 1

L136 596 596 593 595 2 143 148 138 143 5

L137 206 171 175 184 19 36 36 29 34 4

L139 472 445 474 464 16 113 115 116 115 2

L140

L144

L146 407 409 405 407 2 98 99 98 98 1

L149 390 389 392 390 2 94 94 95 94 1

L151

L152 558 577 575 570 10 111 113 114 113 2

L155

L157

L158 427 424 424 425 2 428 424 423 425 3

L159

L160 410 420 418 416 5 99 101 99 100 1

L164

L165 500 498 489 496 6 112 112 111 112 1

L168 434 440 438 437 3 110 113 112 112 2

L170 437 445 439 440 4 104 103 104 104 1

L171

L172

L176

L177

L179 483 467 470 62 57 71

L182 430 430 434 431 2 101 103 101 102 1

 Consensus Mean 448 Consensus Mean 109

 Consensus Standard Deviation 63 Consensus Standard Deviation 31

 Maximum 595 Maximum 493

 Minimum 184 Minimum 34

 N 22 N 21

Individual Results

DHA

SRM 3275-I Fish Oil (mg/g) SRM 3275-III Fish Oil (mg/g)

Community 

Results
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Figure 19. Linoleic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation and 

analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST certified value bounded by twice its uncertainty (U95).
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Figure 20. Linoleic acid in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST certified value bounded by twice its uncertainty (U95).
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Figure 21. α-Linolenic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
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Figure 22. α-Linolenic acid in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
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Figure 23. γ-Linolenic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
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Figure 24. γ-Linolenic acid in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
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Figure 25. Arachidonic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
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Figure 26. Arachidonic acid in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. No NIST-determined value is available for this sample.
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Figure 27. EPA in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation and analytical 

method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The 

color of the data point represents the sample preparation procedure and analytical method employed. The black solid line represents the 

consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and below the consensus 

mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, 

which encompasses the NIST certified value bounded by twice its uncertainty (U95).
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Figure 28. EPA in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation and 

analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
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Figure 29. DHA in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation and analytical 

method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation (rectangle). The 

color of the data point represents the sample preparation procedure and analytical method employed. The black solid line represents the 

consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and below the consensus 

mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for “acceptable” performance, 

which encompasses the NIST reference value bounded by twice its uncertainty (U95).
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Figure 30. DHA in SRM 3275-III Omega-3 and Omega-6 Fatty Acids in Fish Oil (data summary view – sample preparation and 

analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
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Figure 31. Linoleic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil and SRM 3275-III Omega-3 and Omega-6 

Fatty Acids in Fish Oil (sample/sample comparison view). In this view, the individual laboratory results for one sample (fish oil 1) are 

compared to the results for a second sample (fish oil 2). The solid red box represents the target zone for the two samples, fish oil 1 (xaxis) and fish oil 2 (y-axis). The dotted blue box represents the consensus zone for fish oil 1 (x-axis) and fish oil 2 (y-axis).
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Figure 32. α-Linolenic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil and SRM 3275-III Omega-3 and Omega-6 

Fatty Acids in Fish Oil (sample/sample comparison view). In this view, the individual laboratory results for one sample (fish oil 1) are 

compared to the results for a second sample (fish oil 2). The solid red box represents the target zone for the two samples, fish oil 1 (xaxis) and fish oil 2 (y-axis). The dotted blue box represents the consensus zone for fish oil 1 (x-axis) and fish oil 2 (y-axis).
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Figure 33. γ-Linolenic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil and SRM 3275-III Omega-3 and Omega-6 

Fatty Acids in Fish Oil (sample/sample comparison view). In this view, the individual laboratory results for one sample (fish oil 1) are 

compared to the results for a second sample (fish oil 2). The solid red box represents the target zone for the two samples, fish oil 1 (xaxis) and fish oil 2 (y-axis). The dotted blue box represents the consensus zone for fish oil 1 (x-axis) and fish oil 2 (y-axis).
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Figure 34. Arachidonic acid in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil and SRM 3275-III Omega-3 and Omega6 Fatty Acids in Fish Oil (sample/sample comparison view). In this view, the individual laboratory results for one sample (fish oil 1) 

are compared to the results for a second sample (fish oil 2). The dotted blue box represents the consensus zone for fish oil 1 (x-axis) 

and fish oil 2 (y-axis).
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Figure 35. EPA in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil and SRM 3275-III Omega-3 and Omega-6 Fatty Acids 

in Fish Oil (sample/sample comparison view). In this view, the individual laboratory results for one sample (fish oil 1) are compared to 

the results for a second sample (fish oil 2). The solid red box represents the target zone for the two samples, fish oil 1 (x-axis) and fish 

oil 2 (y-axis). The dotted blue box represents the consensus zone for fish oil 1 (x-axis) and fish oil 2 (y-axis).
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Figure 36. DHA in SRM 3275-I Omega-3 and Omega-6 Fatty Acids in Fish Oil and SRM 3275-III Omega-3 and Omega-6 Fatty Acids 

in Fish Oil (sample/sample comparison view). In this view, the individual laboratory results for one sample (fish oil 1) are compared to 

the results for a second sample (fish oil 2). The solid red box represents the target zone for the two samples, fish oil 1 (x-axis) and fish 

oil 2 (y-axis). The dotted blue box represents the consensus zone for fish oil 1 (x-axis) and fish oil 2 (y-axis).
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CHLOROGENIC ACID, FLAVONOIDS, AND NAPHTHODIANTHRONES IN ST. 

JOHN’S WORT DIETARY SUPPLEMENTS

Study Overview

In this study, participants were provided with two NIST SRMs, SRM 3262 St. John’s Wort

(Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort (Hypericum perforatum 

L.) Methanol Extract. Participants were asked to use in-house analytical methods to determine the 

mass fractions of chlorogenic acid, rutin, hyperoside, isoquercitrin, quercitrin, quercetin, 

amentoflavone, pseudohypericin, and hypericin in each of the matrices and report values on an asreceived basis.

Sample Information

St. John’s Wort Aerial Parts. Participants were provided with three packets containing 

approximately 3.3 g of dried St. John’s Wort aerial parts. The dried leaves were ground, 

homogenized, and packaged inside 4 mil polyethylene bags, which were then sealed inside 

nitrogen-flushed aluminized plastic bags along with two packets of silica gel. Before use, 

participants were instructed to thoroughly mix the contents of each packet and use a sample size 

of at least 1.0 g. Participants were asked to store the material at controlled room temperature, 

10 °C to 30 °C, and to report a single value from each packet provided. Approximate analyte 

levels were not reported to participants prior to the study. The target values for chlorogenic acid, 

rutin, hyperoside, quercitrin, pseudohypericin, and hypericin in SRM 3262 St. John’s Wort

(Hypericum perforatum L.) Aerial Parts were determined at NIST using liquid chromatography 

with absorbance and fluorescence detection following Soxhlet extraction. Target values for 

amentoflavone, isoquercitrin, and quercetin have not been established in this material. The NISTdetermined values and uncertainties for chlorogenic acid, rutin, hyperoside, quercitrin, 

pseudohypericin, and hypericin are provided in the table below, on an as-received basis.

NIST-Determined Mass Fraction in SRM 3262 (mg/g)

Analyte (as-received basis)

Chlorogenic acid 0.154 ± 0.007

Rutin 5.05 ± 0.11

Hyperoside 5.02 ± 0.11

Quercitrin 0.984 ± 0.030

Pseudohypericin 0.711 ± 0.020

Hypericin 0.515 ± 0.018

St. John’s Wort Methanol Extract. Participants were provided with three packets containing 

approximately 1.6 g of St. John’s Wort methanol extract. The extract was ground, homogenized, 

and packaged inside 4 mil polyethylene bags, which were then sealed inside nitrogen-flushed 

aluminized plastic bags along with two packets of silica gel. Before use, participants were 

instructed to thoroughly mix the contents of each packet and use a sample size of at least 0.1 g. 

Participants were asked to store the material at controlled room temperature, 10 °C to 30 °C, and 

to report a single value from each packet provided. Approximate analyte levels were not reported 

to participants prior to the study. The reference values for chlorogenic acid, rutin, hyperoside, 
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isoquercitrin, quercitrin, pseudohypericin, and hypericin in SRM 3264 St. John’s Wort (Hypericum 

perforatum L.) Methanol Extract were determined at NIST using liquid chromatography with 

absorbance and fluorescence detection following Soxhlet extraction. Target values for 

amentoflavone and quercetin have not been established in this material. The NIST-determined 

values and uncertainties for chlorogenic acid, rutin, hyperoside, isoquercitrin, quercitrin, 

pseudohypericin, and hypericin are provided in the table below, both on a dry-mass basis and on 

an as-received basis accounting for moisture of the material (0.92 %).

Reference Mass Fraction in SRM 3264 (mg/g)

Analyte (dry-mass basis) (as-received basis)

Chlorogenic acid 1.050 ± 0.059 1.040 ± 0.058

Rutin 34.3 ± 1.7 34.0 ± 1.7

Hyperoside 17.66 ± 0.88 17.50 ± 0.87

Isoquercitrin 9.47 ± 0.46 9.38 ± 0.46

Quercitrin 3.23 ± 0.16 3.20 ± 0.16

Pseudohypericin 0.809 ± 0.031 0.802 ± 0.031

Hypericin 0.439 ± 0.017 0.435 ± 0.017

Study Results

• Thirty-nine laboratories enrolled in this exercise and received samples. Seventeen 

laboratories reported data for at least one analyte in the St. John’s wort samples (44 % 

participation).

• The consensus means for rutin in the St. John’s wort extract and quercitrin in the St. John’s 

wort aerial parts were within the target ranges with acceptable between-laboratory 

variability (14 % and 20 % RSD, respectively).

• The consensus means were above the target ranges for chlorogenic acid and hypericin in 

both samples, as well as for hyperoside, pseudohypericin, and quercitrin in the St. John’s 

wort extract.

• Observed between-laboratory variability was excellent for chlorogenic acid, 

hyperoside, and quercitrin in the St. John’s wort extract (6 % to 14 % RSD).

• Between-laboratory variability was extremely high for chlorogenic acid in the St. 

John’s wort aerial parts, for pseudohypericin in St. John’s wort extract, and hypericin

in both matrices (55 % to 97 % RSD).

• The consensus means were below the target ranges for rutin, hyperoside, and

pseudohypericin, in the St. John’s wort aerial parts, and for isoquercitrin in the St. John’s 

wort extract.

• Observed between-laboratory variability was excellent for isoquercitrin in the St. 

John’s wort extract (10 % RSD).

• Between-laboratory variability was acceptable for rutin and hyperoside in the St. John’s 

wort aerial parts (23 % to 28 % RSD).

• Between-laboratory variability was extremely high for pseudohypericin in St. John’s 

wort aerial parts (85 % RSD).
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• A majority of the laboratories reported using solvent extraction as the sample preparation 

method (88 %). One laboratory reported using open beaker digestion a sample preparation 

technique (6 %), and one laboratory reported that no sample preparation was used (6 %).

• A majority of the laboratories reported using LC-absorbance as the analytical approach 

(82 %). One laboratory reported using UV-VIS (6 %) as their instrumental method, and 

one laboratory reported using HPLC (6 %).

Technical Recommendations

The following recommendations are based on results obtained from the participants in this study. 

• With a small number of laboratories reporting data for these compounds, and a majority 

reporting use of the same or very similar methods, drawing extensive technical 

conclusions is difficult.

• No methods presented as significantly better or worse than any other. No systematic biases 

were noted.

• Some laboratories using LC-absorbance may be experiencing a co-elution that would cause 

a high bias in the results. The problem can likely be corrected by alteration of the 

chromatographic conditions. The following recommendations can help identify and avoid 

potential coelutions.

• A chromatographic method with alternate selectivity (different retention order) can be 

used as a check for each new sample type that is run. Ideally, the retention of coeluting 

compounds would also be affected and the results from the two chromatographic 

systems would be different. Two different responses would indicate a possible bias in 

one approach.

• A different detector can be used in series with an absorbance detector (as confirmation), 

such as a fluorescence detector or mass spectrometer. If a coeluting compound is 

present, the response from these detectors would be different than the response from 

the absorbance detector. Two different responses would indicate a possible bias in one 

approach.

• Considerations of potential interferences can assist in troubleshooting. Understanding 

the matrix that is being tested and possible coeluting compounds can be evaluated 

before a sample is analyzed for additional confidence in the result.

• Low results for some compounds (such as rutin, isoquercitrin, and hyperoside) may be the 

result of an incomplete extraction, or only partial hydrolysis of glycosides.

• Calibration materials had a lower purity than expected. It is important to critically evaluate 

the purity of standards.
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Table 18. Individualized data summary table (NIST) for chlorogenic acid, flavonoids, and naphthodianthrones in St. John’s wort dietary 

supplements.

Lab Code: NIST

Analyte Sample Units xi si Z'comm ZNIST N x* s* xNIST U 95

Chlorogenic Acid SJW Aerial Parts mg/g 0.154 0.015 0.00 8 0.186 0.130 0.154 0.015

Chlorogenic Acid SJW Extract mg/g 1.04 0.12 0.00 9 1.26 0.17 1.04 0.12

Rutin SJW Aerial Parts mg/g 5.05 0.22 0.00 17 3.57 0.84 5.05 0.22

Rutin SJW Extract mg/g 34.0 3.4 0.00 17 30.9 4.5 34.0 3.4

Hyperoside SJW Aerial Parts mg/g 5.02 0.22 0.00 11 3.48 0.98 5.02 0.22

Hyperoside SJW Extract mg/g 17.5 1.7 0.00 11 20.3 1.2 17.5 1.7

Isoquercitrin SJW Aerial Parts mg/g 8 1.44 0.37

Isoquercitrin SJW Extract mg/g 9.38 0.91 0.00 8 7.58 0.72 9.38 0.91

Quercitrin SJW Aerial Parts mg/g 0.984 0.060 0.00 10 0.952 0.191 0.984 0.060

Quercitrin SJW Extract mg/g 3.20 0.32 0.00 10 3.94 0.43 3.20 0.32

Quercetin SJW Aerial Parts mg/g 16 2.01 0.32

Quercetin SJW Extract mg/g 16 6.44 0.80

Amentoflavone SJW Aerial Parts mg/g 2 0.0370 0.0040

Amentoflavone SJW Extract mg/g 2 0.0980 0.0040

Pseudohypericin SJW Aerial Parts mg/g 0.711 0.040 0.00 6 0.605 0.514 0.711 0.040

Pseudohypericin SJW Extract mg/g 0.802 0.061 0.00 6 1.310 0.726 0.802 0.061

Hypericin SJW Aerial Parts mg/g 0.515 0.036 0.00 7 0.781 0.676 0.515 0.036

Hypericin SJW Extract mg/g 0.435 0.034 0.00 8 1.609 1.560 0.435 0.034

xi Mean of reported values N Number of quantitative xNIST NIST-assessed value

si Standard deviation of reported values values reported U95 ±95% confidence interval

Z'comm Z'-score with respect to community x* Robust mean of reported about the assessed value or

 consensus values standard deviation (sNIST)

ZNIST Z-score with respect to NIST value s* Robust standard deviation

National Institute of Standards & Technology

Exercise L - October 2015 - Botanical Analytes

1. Your Results 2. Community Results 3. Target
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Table 19. Data summary table for chlorogenic acid in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 0.154 0.015 1.04 0.12

L101

L102 0.760 0.820 0.630 0.737 0.097 3.47 3.38 3.54 3.46 0.08

L103 < 0.100 < 0.100 < 0.100 1.04 1.10 1.09 1.08 0.03

L104

L105

L107

L110 0.245 0.246 0.246 0.246 0.001 1.26 1.33 1.31 1.30 0.04

L111 0.225 0.224 0.223 0.224 0.001 1.24 1.31 1.31 1.29 0.04

L112

L113

L118

L120

L122

L125 0.188 0.191 0.207 0.195 0.010 1.30 1.29 1.29 1.29 0.01

L126

L128 0.140 0.110 0.130 0.127 0.015 1.38 1.36 1.38 1.37 0.01

L130

L131

L133

L137

L138 0.080 0.080 0.070 0.077 0.006 1.24 1.25 1.26 1.25 0.01

L139

L141

L144 0.010 0.010 0.010 0.000 0.80 0.70 0.75 0.07

L150

L151

L153

L155

L157

L159

L160

L163

L164

L165 0.230 0.230 0.230 0.230 0.000 1.24 1.24 1.25 1.24 0.01

L166

L170

L172

L177

L179

 Consensus Mean 0.186 Consensus Mean 1.26

 Consensus Standard Deviation 0.130 Consensus Standard Deviation 0.17

 Maximum 0.737 Maximum 3.46

 Minimum 0.010 Minimum 0.75

 N 8 N 9

Individual Results Community Results

Chlorogenic Acid

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)
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Table 20. Data summary table for rutin in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 5.05 0.22 34.0 3.4

L101

L102 5.68 5.98 6.35 6.00 0.34 8.3 9.3 9.7 9.1 0.7

L103

L104

L105

L107

L110 5.46 5.53 5.43 5.47 0.05 32.3 35.2 33.4 33.6 1.5

L111 3.15 3.05 2.87 3.02 0.14 29.1 29.2 29.5 29.3 0.2

L112

L113

L118

L120 4.21 3.70 3.69 3.87 0.30 33.1 33.6 33.0 33.2 0.3

L122

L126 3.60 3.32 3.94 3.62 0.31 34.2 34.0 33.9 34.0 0.2

L128

L130

L131

L133 2.31 2.20 2.11 2.21 0.10 29.1 27.9 29.4 28.8 0.8

L137 50.25 46.97 48.28 48.50 1.65 249.9 241.5 253.5 248.3 6.2

L138 3.23 3.25 3.14 3.21 0.06 31.1 30.9 31.2 31.1 0.1

L139 3.25 3.82 3.31 3.46 0.31 14.9 15.5 14.5 15.0 0.5

L140 3.75 4.26 3.40 3.80 0.43 33.9 33.3 33.7 33.6 0.3

L141 3.27 3.51 3.67 3.48 0.20 33.8 33.3 32.4 33.2 0.7

L144 3.80 3.50 3.65 0.21 33.4 33.5 33.5 0.1

L150

L151 3.32 3.64 3.75 3.57 0.22 34.6 34.8 34.8 34.8 0.1

L153

L155

L157 2.76 3.62 3.43 3.27 0.45 31.2 31.9 31.0 31.3 0.5

L159

L160

L163

L164

L165 1.60 1.70 1.68 1.66 0.05 15.7 16.0 15.9 15.9 0.2

L166

L170

L171 3.44 3.45 3.52 3.47 0.04 29.7 30.6 29.2 29.8 0.7

L172

L177

L179 3.25 3.21 3.24 3.23 0.02 29.0 29.1 29.0 29.0 0.1

 Consensus Mean 3.57 Consensus Mean 30.9

 Consensus Standard Deviation 0.84 Consensus Standard Deviation 4.5

 Maximum 48.50 Maximum 248.3

 Minimum 1.66 Minimum 9.1

 N 17 N 17

Rutin

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)

Community 

Results Individual Results
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Table 21. Data summary table for hyperoside in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 5.05 0.22 34.0 3.4

L101

L102 5.68 5.98 6.35 6.00 0.34 8.3 9.3 9.7 9.1 0.7

L103

L104

L105

L107

L110 5.46 5.53 5.43 5.47 0.05 32.3 35.2 33.4 33.6 1.5

L111 3.15 3.05 2.87 3.02 0.14 29.1 29.2 29.5 29.3 0.2

L112

L113

L118

L120 4.21 3.70 3.69 3.87 0.30 33.1 33.6 33.0 33.2 0.3

L122

L126 3.60 3.32 3.94 3.62 0.31 34.2 34.0 33.9 34.0 0.2

L128

L130

L131

L133 2.31 2.20 2.11 2.21 0.10 29.1 27.9 29.4 28.8 0.8

L137 50.25 46.97 48.28 48.50 1.65 249.9 241.5 253.5 248.3 6.2

L138 3.23 3.25 3.14 3.21 0.06 31.1 30.9 31.2 31.1 0.1

L139 3.25 3.82 3.31 3.46 0.31 14.9 15.5 14.5 15.0 0.5

L140 3.75 4.26 3.40 3.80 0.43 33.9 33.3 33.7 33.6 0.3

L141 3.27 3.51 3.67 3.48 0.20 33.8 33.3 32.4 33.2 0.7

L144 3.80 3.50 3.65 0.21 33.4 33.5 33.5 0.1

L150

L151 3.32 3.64 3.75 3.57 0.22 34.6 34.8 34.8 34.8 0.1

L153

L155

L157 2.76 3.62 3.43 3.27 0.45 31.2 31.9 31.0 31.3 0.5

L159

L160

L163

L164

L165 1.60 1.70 1.68 1.66 0.05 15.7 16.0 15.9 15.9 0.2

L166

L170

L171 3.44 3.45 3.52 3.47 0.04 29.7 30.6 29.2 29.8 0.7

L172

L177

L179 3.25 3.21 3.24 3.23 0.02 29.0 29.1 29.0 29.0 0.1

 Consensus Mean 3.57 Consensus Mean 30.9

 Consensus Standard Deviation 0.84 Consensus Standard Deviation 4.5

 Maximum 48.50 Maximum 248.3

 Minimum 1.66 Minimum 9.1

 N 17 N 17

Rutin

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)

Community 

Results Individual Results
80

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Table 22. Data summary table for isoquercitrin in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 9.38 0.91

L101

L103

L104

L105

L107

L110 2.43 2.45 2.41 2.43 0.02 7.85 8.56 8.04 8.15 0.37

L112

L113

L120 1.58 1.40 1.34 1.44 0.12 7.12 7.59 7.43 7.38 0.24

L122

L126 1.33 1.24 1.06 1.21 0.13 6.43 6.49 6.36 6.42 0.06

L128

L130

L131

L137

L138 1.83 1.85 1.77 1.82 0.04 12.09 12.03 12.15 12.09 0.06

L139

L140 1.47 1.39 1.52 1.46 0.07 7.49 7.61 7.99 7.70 0.26

L141 1.17 1.31 1.39 1.29 0.11 7.73 7.46 7.43 7.54 0.16

L151 0.99 1.00 1.01 1.00 0.01 7.33 7.34 7.42 7.36 0.05

L153

L155

L157 1.02 1.44 1.37 1.28 0.22 7.34 7.44 7.27 7.35 0.09

L159

L163

L164

L166

L170

L172

L177

L179

 Consensus Mean 1.44 Consensus Mean 7.58

 Consensus Standard Deviation 0.37 Consensus Standard Deviation 0.72

 Maximum 2.43 Maximum 12.09

 Minimum 1.00 Minimum 6.42

 N 8 N 8

Isoquercitrin

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)

Community 

Results Individual Results
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Table 23. Data summary table for quercitrin in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 0.984 0.060 3.20 0.32

L101

L103

L104

L105

L107

L110 1.210 1.220 1.190 1.207 0.015 3.59 3.90 3.70 3.73 0.16

L112

L113

L120 1.022 0.951 0.962 0.978 0.038 3.75 3.81 3.84 3.80 0.05

L122

L126 1.056 0.987 0.936 0.993 0.060 3.91 3.88 3.93 3.91 0.03

L128

L130

L131

L137 0.710 0.687 0.733 0.710 0.023 3.40 3.44 3.33 3.39 0.06

L138 0.928 0.931 0.906 0.922 0.014 3.63 3.61 3.63 3.62 0.01

L139

L140 0.800 0.740 0.820 0.787 0.042 5.44 5.80 5.66 5.63 0.18

L141 0.899 0.928 0.952 0.926 0.027 3.99 3.89 3.93 3.94 0.05

L144 2.100 1.900 2.000 0.141 6.20 6.20 6.20 0.00

L151 0.837 0.824 0.827 0.829 0.007 3.88 4.03 3.95 3.95 0.08

L153

L155

L157 0.823 1.003 0.972 0.933 0.096 3.90 3.89 3.84 3.88 0.03

L159

L163

L164

L166

L170

L172

L177

L179

 Consensus Mean 0.952 Consensus Mean 3.94

 Consensus Standard Deviation 0.191 Consensus Standard Deviation 0.43

 Maximum 2.000 Maximum 6.20

 Minimum 0.710 Minimum 3.39

 N 10 N 10

Quercitrin

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)

Individual Results Community Results
82

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Table 24. Data summary table for quercetin in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST

L101

L102 2.07 2.15 2.25 2.16 0.09 5.96 5.99 6.06 6.00 0.05

L103 23.90 24.30 24.80 24.33 0.45 98.00 98.60 94.50 97.03 2.21

L104

L105

L107

L110 2.38 2.38 2.35 2.37 0.02 6.09 6.59 6.27 6.32 0.25

L111 1.93 1.83 1.78 1.85 0.08 5.43 5.45 5.51 5.46 0.04

L112

L113

L118

L120 1.80 1.79 1.77 1.78 0.02 6.23 6.35 6.23 6.27 0.07

L122

L126 1.98 1.96 2.00 1.98 0.02 7.47 7.25 7.47 7.40 0.13

L128

L130

L131

L133 1.54 1.49 1.43 1.49 0.06 6.07 5.73 5.81 5.87 0.18

L134

L137 1.92 1.88 1.94 1.91 0.03 5.63 5.58 5.55 5.59 0.04

L138 2.14 2.16 2.09 2.13 0.04 6.34 6.27 6.33 6.31 0.04

L139 2.19 2.30 2.20 2.23 0.06 5.99 6.09 5.85 5.98 0.12

L140 1.89 1.86 1.82 1.86 0.04 6.63 6.38 6.59 6.53 0.13

L141 1.77 1.71 1.71 1.73 0.03 6.80 6.70 6.53 6.68 0.14

L144

L150

L151 1.97 1.94 1.99 1.97 0.02 7.47 7.54 7.47 7.49 0.04

L153

L155

L157 1.54 1.63 1.60 1.59 0.04 5.89 5.96 5.73 5.86 0.12

L159

L160

L163

L164

L165 2.12 2.11 2.10 2.11 0.01 6.08 6.23 6.16 6.16 0.08

L166

L170

L171 1.91 1.92 2.00 6.33 6.38 6.37

L172

L177

L179 28.29 28.03 28.23 28.18 0.14 105.01 108.92 103.23 105.72 2.91

 Consensus Mean 2.01 Consensus Mean 6.44

 Consensus Standard Deviation 0.32 Consensus Standard Deviation 0.80

 Maximum 28.18 Maximum 105.72

 Minimum 1.49 Minimum 5.46

 N 16 N 16

Quercetin

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)

Community 

Results Individual Results
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Table 25. Data summary table for amentoflavone in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST

L103

L104

L105

L107

L110 0.0410 0.0380 0.0370 0.0387 0.0021 0.0930 0.0990 0.0960 0.0960 0.0030

L112

L120

L122

L126

L128

L130

L137

L138 0.0340 0.0350 0.0340 0.0343 0.0006 0.1050 0.0990 0.0950 0.0997 0.0050

L139

L141

L151

L153

L155

L157

L159

L163

L164

L166

L170

L172

L177

L179

 Consensus Mean 0.0370 Consensus Mean 0.0980

 Consensus Standard Deviation 0.0040 Consensus Standard Deviation 0.0040

 Maximum 0.0387 Maximum 0.0997

 Minimum 0.0343 Minimum 0.0960

 N 2 N 2

Amentoflavone

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)

Community 

Results Individual Results
84

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Table 26. Data summary table for pseudohypericin in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 0.711 0.040 0.802 0.061

L102 0.360 0.360 0.360 0.360 0.000 1.760 1.770 1.760 1.763 0.006

L103

L104

L105

L107

L110 1.090 1.050 1.090 1.077 0.023 1.230 1.210 1.230 1.223 0.012

L111 1.080 1.070 1.040 1.063 0.021 1.460 1.480 1.480 1.473 0.012

L112

L120

L122

L126

L128

L130

L131

L133

L137 0.875 0.885 0.872 0.877 0.007 2.067 2.250 2.199 2.172 0.094

L138

L139 0.081 0.082 0.084 0.082 0.002 0.810 0.850 0.840 0.833 0.021

L141

L144

L151

L153

L155

L157

L159

L163

L164

L165 0.134 0.206 0.179 0.173 0.036 0.415 0.360 0.412 0.396 0.031

L166

L170

L172

L177

L179

 Consensus Mean 0.605 Consensus Mean 1.310

 Consensus Standard Deviation 0.514 Consensus Standard Deviation 0.726

 Maximum 1.077 Maximum 2.172

 Minimum 0.082 Minimum 0.396

 N 6 N 6

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)

Pseudohypericin

Community 

Results Individual Results
85

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Table 27. Data summary table for hypericin in St. John’s wort dietary supplements.

Lab A B C Avg SD A B C Avg SD

NIST 0.515 0.036 0.435 0.034

L102 1.460 1.290 1.340 1.363 0.087 1.550 1.570 1.560 1.560 0.010

L103 5.600 4.900 5.500 5.333 0.379

L104

L105

L107

L110 0.453 0.435 0.454 0.447 0.011 0.390 0.397 0.401 0.396 0.006

L111 0.607 0.592 0.585 0.595 0.011 0.603 0.676 0.636 0.638 0.037

L112

L118

L120

L122

L124

L126

L128

L130

L131

L133

L134

L137 0.767 0.719 0.730 0.739 0.025 1.756 1.859 1.786 1.800 0.053

L138

L139 0.008 0.008 0.009 0.008 0.001 0.170 0.180 0.150 0.167 0.015

L141

L144

L150

L151

L153

L155

L157

L159

L163

L164

L165 0.536 0.528 0.553 0.539 0.013 1.053 1.003 1.080 1.045 0.039

L166

L170 1.810 1.770 1.750 1.777 0.031 3.310 3.330 3.310 3.317 0.012

L172

L177

L179

 Consensus Mean 0.781 Consensus Mean 1.609

 Consensus Standard Deviation 0.676 Consensus Standard Deviation 1.560

 Maximum 1.777 Maximum 5.333

 Minimum 0.008 Minimum 0.167

 N 7 N 8

Hypericin

SRM 3262 St. John's Wort Aerial Parts (mg/g) SRM 3264 St. John's Wort Extract (mg/g)

Community 

Results Individual Results
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Figure 37. Chlorogenic acid in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
87

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Figure 38. Chlorogenic acid in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
88

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Figure 39. Rutin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample preparation and 

analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
89

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Figure 40. Rutin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
90

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Figure 41. Hyperoside in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
91

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Figure 42. Hyperoside in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
92

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Figure 43. Isoquercitrin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. No NIST-determined value is available for this 

sample.
93

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Figure 44. Isoquercitrin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
94

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Figure 45. Quercitrin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
95

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Figure 46. Quercitrin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
96

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Figure 47. Quercetin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. No NIST-determined value is available for this sample.
97

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Figure 48. Quercetin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. No NIST-determined value is available for this 

sample.
98

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Figure 49. Amentoflavone in St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample preparation and 

analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. No NIST-determined value is available for this sample.
99

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Figure 50. Amentoflavone in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. No NIST-determined value is available for this 

sample.
100

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Figure 51. Pseudohypericin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
101

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Figure 52. Pseudohypericin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
102

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Figure 53. Hypericin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts (data summary view – sample preparation

and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard deviation 

(rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The black solid 

line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values above and 

below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone for 

“acceptable” performance, which encompasses the NIST-determined value bounded by twice its uncertainty (U95).
103

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Figure 54. Hypericin in SRM 3264 St. John’s Wort (Hypericum perforatum L.) Methanol Extract (data summary view – sample 

preparation and analytical method). In this view, individual laboratory data are plotted (circles) with the individual laboratory standard 

deviation (rectangle). The color of the data point represents the sample preparation procedure and analytical method employed. The 

black solid line represents the consensus mean, and the green shaded region represents the range of tolerance, calculated as the values 

above and below the consensus mean that result in an acceptable Z’ score, |ZZ′| ≤ 2. The red shaded region represents the target zone 

for “acceptable” performance, which encompasses the NIST reference value bounded by twice its uncertainty (U95).
104

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Figure 55. Chlorogenic acid in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort

(Hypericum perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one 

sample (St. John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The solid red 

box represents the target zone for the two samples, St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis). 

The dotted blue box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
105

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Figure 56. Rutin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort (Hypericum 

perforatum L.) (sample/sample comparison view). In this view, the individual laboratory results for one sample (St. John’s wort aerial 

parts) are compared to the results for a second sample (St. John’s wort methanol extract). The solid red box represents the target zone 

for the two samples, St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis). The dotted blue box represents 

the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
106

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Figure 57. Hyperoside in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort

(Hypericum perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one 

sample (St. John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The solid red 

box represents the target zone for the two samples, St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis). 

The dotted blue box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
107

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Figure 58. Isoquercitrin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort

(Hypericum perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one 

sample (St. John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The dotted blue 

box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
108

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Figure 59. Quercitrin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort (Hypericum 

perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one sample (St. 

John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The solid red box represents 

the target zone for the two samples, St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis). The dotted blue 

box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
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Figure 60. Quercetin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort (Hypericum 

perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one sample (St. 

John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The dotted blue box 

represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
110

This publication is available free of charge from: https://doi.org/10.6028/NIST.IR.8154

Figure 61. Amentoflavone in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort

(Hypericum perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one 

sample (St. John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The dotted blue 

box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
111

This publication is available free of charge from: https://doi.org/10.6028/NIST.IR.8154

Figure 62. Pseudohypericin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort

(Hypericum perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one 

sample (St. John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The solid red 

box represents the target zone for the two samples, St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis). 

The dotted blue box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).
112

This publication is available free of charge from: https://doi.org/10.6028/NIST.IR.8154

Figure 63. Hypericin in SRM 3262 St. John’s Wort (Hypericum perforatum L.) Aerial Parts and SRM 3264 St. John’s Wort (Hypericum 

perforatum L.) Methanol Extract (sample/sample comparison view). In this view, the individual laboratory results for one sample (St. 

John’s wort aerial parts) are compared to the results for a second sample (St. John’s wort methanol extract). The solid red box represents 

the target zone for the two samples, St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis). The dotted blue 

box represents the consensus zone for St. John’s wort aerial parts (x-axis) and St. John’s wort methanol extract (y-axis).